Bioimaging Instructor
Bioimaging Instructor kdorfman Tue, 06/16/2009 - 18:07Here are the preparation procedures for Bioimaging labs.
Bioimaging F 2018
Bioimaging F 2018 kdorfman Tue, 07/31/2012 - 15:51Cells for lab
day | date | Lab | cells | stained for |
---|---|---|---|---|
W | 9/5 | 1. intro | fixed LL | phalloidin or MT, DAPI |
F | 9/7 | 2. Image formation | fixed LL | phalloidin or MT, DAPI |
W | 9/12 | 3. making figures | none | |
F | 9/14 | 4. NA | fixed LL | MT, phalloidin, DAPI |
W | 9/19 | 5. Resolution | fixed LL | MT, DAPI |
F | 9/21 | 6. fluorescence | fixed LL | MT, phalloidin, DAPI REALLY BRIGHT for photobleaching! |
W | 9/26 | lab practical | none | |
F | 9/28 | presentation | none | |
W | 10/3 | 7. immunoflluor | fixed LL , 3t3 (fixed?), live LL on coverslips |
unstained |
F | 10/5 | 8. organelles | fixed LL | Golgi, gamma tub, Hec 1, Lap 2, Alpha actinin, ZO 1 |
F | 10/12 | 9. Live cells |
0: Pre-sem prep
0: Pre-sem prep kdorfman Wed, 12/07/2011 - 15:49Pre-semester prep
Check out 20 minute secondary antibodies from Invitrogen
Get 2 boxes each: 20 mm and 14 mm coverslip dishes from Invitro-Scientific, much cheaper than MatTek.
http://www.invitrosci.com/product_detail.php?product_id=21
http://www.invitrosci.com/product_detail.php?product_id=25
Nucleofector kit: Ingenio Electroporation kit, MIR50112
Fisher: MIR50112 $230.17. More expensive than mirusbio, but mirus charges $35 for shipping.
Solutions
Solutions kdorfman Tue, 12/27/2011 - 19:21Solutions to have at the beginning of the semester
Amount | conc | solution | |
---|---|---|---|
100 | mL | 10% | Tween in water |
50 | mL | 10% | Tween in PBS |
100 | mL | 10% | Triton-X in water |
100 | mL | 10% | Triton-X in PBS |
5 | L | 10X | PBS |
1 | L | 1x | PBS in 10 mL sterile aliquots |
4 | L | 1x | PBS-Tw-Az |
25 | mL | 10% | Sodium azide |
250 | mL | 10X | HBS |
100 | mL | 10 X | Ca-free HBS |
500 | mL | 1x | Non-CO2 medium with serum |
1000 | mL | 1x | Non-CO2 serum free medium |
2 | L | 1x | F10-Hams |
250 | mL | 1x | serum free F-10 Hams |
1 | L | 1x | DMEM |
20 | mL | DAPI Fluoromount Fisher OB010020 2 bottles to share. |
Aliquots
Aliquots kdorfman Tue, 12/27/2011 - 19:27Students use lots of these:
- 10 mL PBS
- 10 mL serum-free-non-CO2 medium
- 10 mL HBSS
- 10 mL HBS
Slides
Slides kdorfman Tue, 12/27/2011 - 20:56slides 2012
slides 2012 kdorfman Fri, 08/23/2013 - 14:07CHECK THIS
LLCPk-1
coverslips, fixed in 3.5% formaldehyde and stored in PBS-tween-azide in coplin jars in the refrigerator:
30 unmounted,
10 mounted, stained only with DAPI
20 stained for tubulin, and mounted with DAPI
20 stained for actin and tubulin, and mounted with DAPI
10 serum starved, stained for tubulin, mounted with DAPI
- for Lab 4.1
- Grow first in regular medium (otherwise they won't stick)
- Replace medium with serum free medium.
- Fix after 24 hours
10 Arrested with 100nM nocodazole (8 - 16 hours)
- for Lab 4.1
- Treat with nocodazole late in the day
- Fix the next day in para-glut or formaldehyde
- Stain for tubulin, mount with DAPI
Nocodazole stock is 33 mM in DMSO
Make 100 µL 1 mM: 3 µL (33mM) + 97 µL medium
Make 1 mL 6µM: 6 µL (1mM) + 994 µL medium
Add 1 drop (~50 µL) 6µM to each dish of ~3 mL
OR
Make 30 mL 100 nM: 3 µL (1mM) in 30 mL medium
Change the medium in each dish.
3t3
12 for Lab 3.3, unmounted
slides 2013
slides 2013 kdorfman Fri, 08/23/2013 - 14:08Lab | date | # | DAPI + | fix |
---|---|---|---|---|
1.1 & 1.3 | 9/4 & 11 | 10 | paraglut | |
1.2 | 9/9 | 10 | alpha tub | paraglut |
2.1 & 2.2 | 9/16 | 10 | alpha tub, actin | paraglut |
3.1 | 9/23 | 10 | alpha tub, actin | paraglut |
3.2 | 9/25 | 2 | Golgi | formaldehyde (no glut) |
2 | gamma tubulin | paraglut or methanol | ||
2 | actin | paraglut | ||
2 | alpha actinin | formaldehyde (no glut) | ||
2 | ZO1 | methanol or paraformaldehyde | ||
2 | LAP2 | methanol | ||
2 | vinculin | formaldehyde |
slides 2015
slides 2015 kdorfman Tue, 07/14/2015 - 14:58LL's for student slides
Fix 90 in formaldehyde; 10 in methanol
Plate 102 in 17 6-well plates.
Lab | # coverslips | fix | stain | secondary |
---|---|---|---|---|
1.1 | 10 | form or paraglut | ||
1.2 | 10 | " | ||
1.3 | 10 | " | ||
2.1 | 10 | " | anti-tubulin, phalloidin | goat anti-rat |
2.2 | 10 | " | anti-tubulin | goat anti-rat |
3.1 | 10 | " | anti-tubulin, phalloidin FRESH | goat anti-rat |
3.2 | 3 | form | alpha actinin | anti-mouse IgM |
3.2 | 3 | form | anti Golgi | anti-mouse IgG |
3.2 | 3 | form | vinculin | |
3.2 | 3 | MeOH | anti gamma-tubulin | |
3.2 | 3 | " | anti-Hec1 | |
3.2 | 3 | " | LAP2 | |
3.3 | 20 | form or paraglut | un-mounted for students to stain |
organelle slides
organelle slides kdorfman Tue, 07/14/2015 - 14:59Alpha actinin
Alpha actinin kdorfman Tue, 07/14/2015 - 15:02Stains adhesions and stress fibers
- Monoclonal, BM 75.2
- Fix in Formaldehyde, no glut or meOH
- 1:200
- IgM, use mouse FITC secondary that recognizes IgMs
Gamma tubulin
Gamma tubulin kdorfman Tue, 07/14/2015 - 15:03Stains Centrosomes
- Monoclonal, GTU-88, Sigma T6557
- Fixation: methanol; para/glut
- Dilution 1:100
From Sigma:
General description
γ-Tubulin (48kDa) is a ubiquitous and highly conserved protein within the microtubule organizing centers (MTOCs) in eukaryotic cells. γ-Tubulin is mapped to human chromosome 17q21.2 and codes for a member of the tubulin family. Human TUBG1 transcript is widely expressed in preimplantation embryos and brain. Monoclonal Anti-γ-Tubulin (mouse IgG1 isotype) is derived from the GTU-88 hybridoma produced by the fusion of mouse myeloma cells and splenocytes from an immunized mouse.
Specificity
Monoclonal Anti-γ-Tubulin recognizes an epitope located in the N-terminal amino acids of γ-tubulin (48 kDa). Cross reactivity has been observed with human, bovine, dog, hamster, rat, mouse, chicken, and Xenopus γ-tubulin. The antibody recognizes an epitope located within the N-terminal region of γ-tubulin.
Immunogen
synthetic γ-tubulin peptide, conjugated to KLH
Application
Monoclonal Anti-γ-Tubulin antibody has been used in western blotting,[1] indirect immunofluorescence (IF) and immunofluorescence staining. Monoclonal Anti-γ-Tubulin is also suitable for use in immunochemical applications such as immunoblotting, immunocytochemical staining of cultured cells and in ELISA. Monoclonal Anti-gamma-Tubulin is suitable for use in immunochemical applications such as immunoblotting, immunocytochemical staining of cultured cells, and in ELISA.
Biochem/physiol Actions
γ-Tubulin nucleates microtubule assembly throughout the mammalian cell cycle in vivo. γ-Tubulin binds microtubule minus ends and is responsible for mediating the link between microtubules and the centrosome. It functions as the microtubule nucleator at the MTOC. It binds to the β-tubulin half of the tubulin molecule, thus establishing the polarity of a microtubule, leaving the α-tubulin half exposed at the plus end. γ-Tubulin abundance is less than 1% of the level of either α- or β-tubulin. Overexpression of γ-tubulin is observed in lung cancer. The expression levels of γ-tubulin can be considered as an important prognostic indicator for patients with astrocytomas.
Physical form
The product is provided as ascites fluid with 15 mM sodium azide as a preservative.
Storage and Stability
For continuous use, store at 2-8 °C for up to one month. For extended storage, freeze in working aliquots. Repeated freezing and thawing, or storage in "frostfree" freezers, is not recommended. If slight turbidity occurs upon prolonged storage, clarify the solution by centrifugation before use.
Disclaimer
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Golgi 58K
Golgi 58K kdorfman Tue, 07/14/2015 - 15:04- Sigma G2404, mouse monoclonal
- Formaldehyde (no glut)
- 1:100
Hec1
Hec1 kdorfman Tue, 07/14/2015 - 15:05Stains kinetochores
- Novus biological, recombinant human HEc1, monoclonal
- Fix in methanol
- 1:200
LAP2
LAP2 kdorfman Tue, 07/14/2015 - 15:07Stains nuclear envelope
- Methanol
- 1:100
- BD transduction
- Mouse IgG
Vinculin
Vinculin kdorfman Tue, 07/14/2015 - 15:08Focal adhesion plaques
- Monoclonal, sigma V4505
- Formaldehyde
- 1:100
2016 schedule
2016 schedule kdorfman Wed, 09/21/2016 - 20:06Lab 1 1 DAPI slide per pair
Lab 2
same DAPI slide as Lab 1
1 H&E slide from histology
Lab 3 Numerical aperture . same DAPI slide
Lab 4 Resolution.
1 slide newly stained phalloidin actin. Must be bright
1 slide fluorescent beads(PS-Speck Microscope Point Source Kit P7220 from Molecular Probes)
Lab 5 Fluorescence. 1 new, bright 3-color stained slide (DAPI, Phalloidin-actin, FITC microtubules)
Lab 6 Dry lab
Lab 7 Labeling Cells 9/28/16
3 fixed LL coverslips
1 3t3 fixed coverslip
1 live LL coverslip for students to fix
paraglut
forceps, mounting medium, pasteur pipettes
Lab 8 - Organelles 10/3/16
Golgi - anti-58K, mouse, formaldehyde fixation
Kinetochore - anti Hec1 - MeOH fixation 1:200 mouse
ER anti-calnexin, paraformaldehyde fixation 1:200
centrosome - anti gamma-tubulin, paraglut, mouse 1:100
nuclear envelope anti LAP2 - MeOH fixation 1:100 mouse
lysosomes
alpha actinin - mouse 1:200 formaldehyde fixation
Lab 8 Practical (and pipet practice)- 10/5
Lab 9 - Imaging Live Cells 10/11 TUESDAY Live cells on mattek
unknown GFP tagged proteins
Thaw GFP lines Wednesday
Split Thursday (~1/4)
Plate Sunday on Mateks for Tuesday
- Thaw
- GFP tubulin
- actin thaw
- EB1 - thaw
- Nucleofect: EGFP-H2B
- split LL parentals on Sunday, HEAVY
- nucleofect & plate Monday by noon
Lab 10 10/12
Single cells to whole organisms
order protists
induce GFP E. coli
CRISPR: Transform guide RNA plasmid into bacteria
Lab 11.1 Motility I 10/17
- thaw B16, 3t3 Thursday 10/13/16 (late in the day)
- plate 9 MatTeks each Fri 10/14/16:
- 3t3
- actin GFP LL
- B16
Lab 11.2 Motility II 10/19
Lab 11.3 Motility III 10/24
Motility presentations 10/26
CRISPR: prepare Cas9/guide RNA plasmid DNA 10/31
CRISPR: prepare repair DNA cassette (PCR, clean up) 11/2
CRISPR: Add Cas9 plasmid + repair DNA to cells 11/7
Lab 13.1 - Cell Signaling 11/9
Lab 13.2 - Cell Signaling 2 11/14
11/16 Wed = Friday schedule
Discussion of independent projects 11/28
Projects 11/30 - 12/14
Final exam period: Final project presentation
2018 cell schedule
2018 cell schedule kdorfman Wed, 12/20/2017 - 18:48Lab | date | number | cell line | live or fixed in | stained with | notes |
---|---|---|---|---|---|---|
1&2 | 1/23 | 10 | LL parental | fixed in paraglut | phalloidin | learn about scope |
3&4 | 1/30 | 10 | LL parental | fixed in paraglut | tubulin, phalloidin, DAPI | NA |
5 | 2/6 | 10 | LL parental (dense) | fixed in paraglut | tubulin, phalloidin, DAPI | photobleaching |
6 | 2/8 | none | Image J, photoshop | |||
7 | 2/13 | 30 | LL parental | fixed in paraglut | unstained | immunofluorescence |
10 | 3t3 | fixed in paraglut | unstained | |||
10 | LL parental | live on coverslip | to fix and stain | |||
8 | 2/15 | Lab practical | ||||
9 | 2/20 | 3 | LL parental | fixed in MeOH | gamma tubulin 1:100 (mouse) (C) | organelle ID lab |
3 | LL parental | fixed in MeOH | Hec1 1:200 (mouse) (didn't do) | |||
3 | LL parental | fixed in MeOH | LAP2 1:200 (mouse) (B) | |||
3 | LL parental | formaldehyde only | Alpha actinin (mouse IgM) 1:200 (D) | |||
3 | LL parental | formaldehyde only | Golgi 58K 1:100 (mouse) (A) | |||
3 | LL parental | doesn't matter | secondary Ab only | |||
9 | 2/22 | 10 | LL a-tub GFP/RFP myosin | live in dish | imaging live cells | |
18 | LL | live in dish | for mitotracker or ceramide | |||
10 | LL H2B m cherry | live in dish | transfected unknowns | |||
10 | 2/27 | GFP E. coli, motile E. coli, Volvox Carolina 152655, Tetrahymena Carolina 131620, Dictyostelium Carolina 55996, Paramecium caudatum Carolina 131554, Chaos (Pelomyxa) carolinensis Carolina 131324 | ||||
11 | 3/1 | 10 | 3t3 | live in dish | motility | |
10 | LL parental | live in dish | ||||
10 | melanoma | live in dish | ||||
10 | LL GFP-actin | live in dish | ||||
12 | 3/6 | 9 | 3t3 | live in dish | Motility II | |
12 | LL GFP-tub | |||||
3 | LL GFP actin | |||||
5 | B16 | |||||
13 | 3/8 | Posters | ||||
14 | 3/20 | 26 | 3t3 | live on coverslip | Endocytosis I | |
3/22 | 18 | 3T3 | live on coverslip | Endocytosis II | ||
10 | LL parental | live on coverslip | ||||
18 | LL a-tub-GFP | live on coverslip | ||||
13 | 3/27 | 28 | 3t3 | live in dish (dense) | Signalling 1 | |
3/29 | 28 | 3t3 | live in dish (dense) | Signalling 2 | ||
14 | 4/3 | 10 | LL-a-tub-GFP | live in dish | mitosis, cytokinesis | |
18 | LL myo tub | live in dish | ||||
15 | 4/5 | 10 | LL- parental | live on coverslip | for fixation | |
10 | LL GFP-tub (or tub & myo) | live in dish | for treatment, observation |
projects 2018
projects 2018 kdorfman Thu, 04/05/2018 - 22:21date | scope | cells | reagents |
---|---|---|---|
4/10 | 1 | 3 LL parentals (coverslip or dish?) | ? |
2 | Volvox | Volvox medium (ordered) | |
3 | LL parentals, dense | ? | |
4 | 4 3t3 live in dish | ? | |
5 | 4 LL GFP tubulin live in dish | ? | |
6 | 3 LL GFP tubulin live in dish? | ? | |
7 | 1 live ll parentals in dish | ceramide | |
8 | 3 GFP actin (in dish?) | cytochalasin D |
date | scope | cells | reagents |
---|---|---|---|
4/12 | 1 | 3 LL parental in dishes | |
2 | Volvox aureus (ordered) | ||
3 | dense LL's (reuse Tuesday's cells? | could use GFP tubulin | |
4 | 6 dishes 3t3 densely plated | ||
5 | 3 live GFP actin LL's, 1 GFP tubulin LL | cytochalasin D, latrunculin | |
6 | 3 GFP tubulin in dish | ||
7 | 2 parental LL in dish | ceramide | |
8 | 3 GFP tubulin in dishes |
date | scope | cells | reagents |
---|---|---|---|
4/19 | 1 | 5 dishes LL parentals, dense | |
2 | Volvox aureus | (will check Monday; order more if needed) | |
3 | will tell us by Tuesday | ||
4 | 5 3t3 in dishes | ||
5 | 3 actin GFP, 3 GFP tubulin LLs (dishes) | ||
6 | 4 GFP tubulin LL's (dishes) | ||
7 | 2 LL parental in dish | NBD ceramide | |
2 GFP-tubulin coverslip | anti-Golgi | ||
8 | 2 GFP tubulin, 1 GFP actin LLs in dishes |
date | scope | cells | reagents |
---|---|---|---|
4/24 | 1 | 3 LL parentals in dish | |
2 | Volvox | ||
3 | 4 dense dishes LL parentals | ||
4 | 3 live 3t3, 3 live LL | ||
5 | 3 live GFP tub LL, 3 actin | ||
6 | 4 GFP tubulin LL live | ||
7 | 3 live LL parentals | ceramide | |
8 | 2 dishes LL GFP tubulin |
2019 F
2019 F kdorfman Thu, 09/12/2019 - 15:46date | prep |
---|---|
9/19 | triple stained LLCPk slides: DAPI, rat anti-alpha tubulin & anti-rat-FITC, red phalloidin |
9/26 | thaw LL flask |
9/27 | change medium |
9/28 | split into large flask |
9/30 | put 5 x 10^5 cells into each of 9 flasks |
10/1 | students nucleofect |
10/3 | Start G418 selection of transfected LLs |
10/9 | Thaw 3t3 into DMEM, spin down, resuspend, plate on coverslips. No adherence (or all dead?) Make new DMEM, replace medium in flasks |
10/10 | Stop selection - cells look bad. Replace selection medium with regular F10Hams |
10/11 | very few adherent cells. |
10/15 | Thaw LL-GFP-alphas,HeLa-H2B-GFP, Mcherry alpha tubulin |
(get HeLa GFP alpha tubulin from Pat) | |
3t3s look fine | |
split transfected cells into one 25 cm2 flask to grow up, one 12.5 cm2 flask to continue selection | |
10/16 | Plate in coverslip dishes for lab on 10/17 (had to be done in Pat's lab) |
10/17 | live cells for mitosis (made in Pat's lab: HeLa-GFP-tubulin, LLCPk-GFP tubulin) |
thaw LLCPk-GFP-actin for use in motility lab 10/22 | |
thaw LLCPk-GFP-tubulin to test freeze/thaw protocol | |
split 3t3s to grow up for live cell observations 10/22 | |
split LL parentals in case anyone wants to repeat nucleofection | |
split nucleofected cells for students; they take over care of them 10/24 | |
10/21 | plate (coverslip bottom dishes) 3t3, LL-GFP-actin from flasks |
make big flask of LL-GFP-actin to freeze | |
thaw b16, spin down, resuspend in DMEM, plate (coverslip dishes) | |
froze 3 vials HeLa-GFP-H2b/mcherry-tubulin | |
10/22 | Bioimaging students split their 2 flasks of nucleofected cells (G418/unselected); made 2 new flasks, 2 coverslip dishes. |
10/23 | Set up sterile hoods with all equipment students will need. |
Freeze 3 vials of 3t3s | |
10/24 | students split nucleofected cells |
10/25 | split LL-GFP-tub, LL-GFP-actin and freeze 2 vials each |
split HeLa-GFP-tub: very strange - some cells never balled up. | |
10/28 | split LL-GFP-tub: 1 flask with 500,000 for nucleofection 10/29, one to keep culture going |
split better looking parentals just to keep going | |
HeLas look OK, but sparse | |
10/29 | students split cells, work on projects |
10/30 | make 1 flask of LL parentals to nucleofect, 1 flask of LL-GFP-tubulin (plus 1 flask each to perpetuate) |
10/31 | students work on their cells |
11/01 | inner incubator door was open, temp was 28C, CO2 was 3 |
replace medium on HeLas (cultures are pretty sparse) | |
replace medium on parentals | |
split LL-GFP-actin to 12.5 flask | |
11/04 | plate 2 coverslip dishes of parental LL's for ER Tracker treatment |
make 2 12.5 flasks for propagation | |
11/05 | |
11/06 | split LL-GFP-tubulin: 2 full flasks for nucleofection, one for propagation |
aliquot 40 mL trypsin | |
freeze 3 vials HeLa-GFP-tubulin | |
one flask HeLa-GFP-tubulin in case students want it | |
split flask of LL-GFP-actin in case students want it | |
11/08 | Kate sick |
11/10 | split parentals |
split LL-GFP-actin | |
refeed HeLa-GFP-tubulin | |
aliquot new F10-Hams & DMEM | |
11/11 | Split BL & AM nucleofected cells: 1 flask for nucleofection, one for propagation |
G418 selection @ 2 mg/mL | |
11/12 | students work on cultures |
11/13 | plate LL-GFP tubulin for nucleofection on 11/14 (with students) |
11/14 | pat brings over IF LL's (intermediate filament eGFP vimentin) |
11/15 | check IF flasks: all light. Split on Monday |
split LLCPK parentals | |
split HeLa-GFP-tubulin | |
11/18 | 8:30 am help students (AZ) count to prep cells (LL-IF-GFP) for nucleofection |
split IF flasks for freezing (2 75cm2 flasks), student projects (2 25 cm2 flasks- each with 500,000 cells in case anyone wants to nucleofect them on Tuesday) | |
11/19 | split LL-GFP-tubulin: 1 heavy, 1 light |
11/20 | split heavy LL-GFP-tubulin, count, put 500,000 in flask for Sam & Brian to nucleofect |
11/21 | students work |
11/22 | LLCPK GFP-IF: freeze 3 (2?) vials from T75 flask; split into T25 flask "in case" |
LLCPK GFP-tubulin: split light | |
J & G: split; will split again 11/29 to make a nucleofection flask 12/1 | |
LLCPK Lifact Actin 488 split into T75 flask for freezing; small flask "in case" | |
KTRM: change medium in dish and flask | |
LLCPK parentals: split lightly "in case" | |
HeLa GFP-Tubulin split lightly "in case" | |
11/25 | ASBK split 2 flasks |
KTRM: | |
GOJC H2B: split | |
Z&A: actin split 1 flask + G418, | |
Z&A IF change medium plut G418 | |
SLBM split GFP-Tub 2 | |
AMBL IF+Actin split plus G418 reduced by half | |
SACW will come in to do their own | |
11/27 | Freeze LifAct-488 LL |
SLBM split "s1" (or wait till 11/29?) | |
SLBM split s2 flask LL-red-mitochondria to a 75 flask to freeze Friday | |
11/29 | LLGFP-GFP-tub split (so students can split on 12/1 for nucleofection 12/2) |
SLBM freeze T75 (get labels from Sam & Brian) | |
HeLas look OK, but sparse |
2021
2021 kdorfman Mon, 07/26/2021 - 16:51Class Schedule:
date | day | cells | reagents |
---|---|---|---|
9/2 | Th | fixed HeLa H2B-GFP mcherry-alpha Tubulin | 10 untreated, and 24 treated with VPA, nocodazole, trichostatin A, cytochalasin D, latrunculin |
9/7 | Tu | ||
9/9 | Th | ||
9/14 | Tu | PS-Speck slides B&G | |
9/16 | Th | ||
9/21 | Tu | photo bleaching HeLa slides | |
9/23 | Th | B16 on coverslips | Para-glut Rat anti-tubulin goat anti-rat PBS-Tw-Az |
9/28 | Tu | 1 flask B16/pair | passaging |
9/30 | Th | view dish from previous lab split for nucleofection |
mini-prep EB1 |
10/5 | Tu | ||
10/7 | Th | ||
10/12 | Tu | ||
10/14 | Th | ||
10/19 | Tu | ||
10/21 | Th | ||
10/26 | Tu | Students split cultures from Drew into 4-chambered dishes | |
10/28 | Th | ||
11/2 | Tu | Students split onto 4 coverslips, 1 flask/pair | 6-well dishes, sterile coverslips |
11/4 | Th | view NLS-GFP cells from Drew's lab, IF | PFA rabbit anti 5mC rabbit anti-DNMT1 goat anti-rabbit green (try red next time) |
11/9 | Tu | ||
11/11 | Th | Veteran's Day | |
11/16 | Tu | ||
11/18 | Th | ||
11/23 | Tu | Th schedule | |
11/25 | Th | Thanksgiving | |
11/30 | Tu | ||
12/2 | Th | ||
12/7 | Tu |
Cell passaging
Cell passaging kdorfman Wed, 09/29/2021 - 17:07Students split a flask of B16s into a new flask and a coverslip-bottom dish for viewing next class
Lab 1 prep
Lab 1 prep kdorfman Wed, 08/25/2021 - 16:09no. | treated with | conc | [stock] | time |
---|---|---|---|---|
10 | untreated | |||
2 | VPA (valproic acid) 1 | 4 uM | 20 uM | over night |
4 | nocodazole 2 | 100 nM | 1 mM (make 1uM 1st) | overnight |
2 | trichostatin A 3 | 200 nM | overnight | |
4 | Cytochalasin D 4 | 250nM | 10 mM | 1 hour |
2 | Latrunculin 5 | 1 uM | 1 mM | 1 hour (in serum free medium!) |
-
Get VPA from Drew. 5X final conc.
Add 0.5625 mL to each well containing 2.25 mL medium. ↩︎ -
Add 97 uL DMSO to the 3 uL in tube to make 100 uL 1mM.
Make 2mL 1uM in medium (2 uL 1mM + 1.998 mL medium).
Add 0.25 mL to the 2.25 mL in each well. ↩︎ -
TSA 10 mM from freezer.
Make 1 mL 10 uM (1 uL TSA + 999 uL medium).
Add 45 uL to each well. ↩︎ -
Add 90 uL DMSO to the tube(makes 1 mM)
Dilute this 1:1000 into medium (makes 1uM) (5X)
Add .256 mL to each well ↩︎ -
Mix 5 uL into 5 mL serum free medium (can use Fluorobrite)
Replace the medium in each well with 2.5 mL of Latrunculin-medium mix ↩︎
2022 (Stephens)
2022 (Stephens) kdorfman Fri, 06/03/2022 - 18:20Pre-semester prep:
6 slides of each cell line (B16 30, 31, 32) all with TRITC phalloidin & DAPI
3 each:
day | date | lab | prep |
---|---|---|---|
Th | 9/29 | image IF slides from previous lab | cell culture demo (9 flasks for students, 18 coverslip bottom dishes for 477H, 8 for Kari |
Tu | 10/4 | live cell observations w/ NucBlue & Mitotracker split into 1 25 cm2 flask for nucleofection |
Eb in liquid culture |
Th | 10/6 | split again, miniprep | |
Tu | 10/11 | nucleofection | aliquot nucleofection reagents |
nucleofection into dish, also flask? | |||
Th | 10/13 | live cell imaging of transfected cells | |
Tu | 10/18 | midterm | |
Th | 10/20 | Plan CURE projects | |
Tu | 10/25 | IF on coverslips: 24 B16 30, 12 B16 31, 12 B16 32 | plate on M 10/24 |
Lamin Ac (mouse) +alpha tubulin (rat), goat anti rat red, goat anti mouse green | fix in paraformaldehyde | ||
Hec1 (mouse) + alpha tubulin (rat), goat anti rat red, goat anti mouse green | humid chambers, colored markers, DAPI, slides |
2023 Fall Stephens
2023 Fall Stephens kdorfman Wed, 07/19/2023 - 14:34day | date | topic | prep |
---|---|---|---|
Tu | 9/5 | Learn your microscope | 10 HeLa parentals, FITC tubulin, 568 phalloidin, DAPI |
Th | 9/7 | Transmitted light and alignment | |
Tu | 9/12 | Cameras and calibration | |
Th | 9/14 | Numerical Aperture and resolution | |
Tu | 9/19 | Fluorescence | |
Th | 9/21 | Fluorescence photobleaching | (10 brand new triple stained HeLa slides?) Drew says no |
Tu | 9/26 | Immunofluorescence procedure | 20 coverslips of fixed HCT116 WT cells (grown on McCoy's) (maybe half treated with a drug the day before fixation), laminA, green secondary alpha tubulin, red secondary |
Th | 9/28 | Immunofluorescence imaging | |
Tu | 10/3 | Live cell imaging and cell culture | Split & seed another dish, live cell staining & imaging nuc blue, mitotracker red |
Th | 10/5 | Plasmid prep. and cell culture | Eb1 in liquid culture, Zymo miniprep |
Tu | 10/10 | Monday schedule | (Kate transfects HCT116's with Eb1 plasmids, seeds on viewing dishes) |
Th | 10/12 | Transfection imaging / Makeup day | image kate-transfected cells |
Tu | 10/17 | Midterm | Mai does trial runs |
Th | 10/19 | FLEXIBLE DAY / CURE intro | |
Tu | 10/24 | 1st day CURE | Students fix & stain coverslips |
Th | 10/26 | ||
Tu | 10/30 | ||
Th | 11/1 | ||
Tu | 11/6 | ||
Th | 11/8 | ||
Tu | 11/13 | ||
Th | 11/15 | ||
Tu | 11/20 | ||
Th | 11/22 | THANKSGIVING | |
Tu | 11/28 | ||
Th | 11/30 | Final Project due | |
Tu | 12/5 | Neurobiology | |
Th | 12/7 | Neurobiology |
09/26/23 Immuno procedure
09/26/23 Immuno procedure kdorfman Thu, 09/21/2023 - 21:19Immunofluorescence Procedure Tu 9/26/23
Materials
- PBS-Tw-Az for rinsing
- Little beakers
- forceps
- enough for 4 humid chambers
- petri dish
- filter paper
- parafilm
- fixed coverslip of HTC116 cells, 1 per student
- Mixed primary antibodies
- Mixed secondary antibodies
- Goat anti mouse 488 (green)
- Goat anti rat TRITC (red)
To mix primaries:
- Lamin A:
- Use 4 aliquots of 5 µL each
- Add 125 µL PBS-Tw-Az-BSA to get (slightly less than) 2X strength
- total volume: 520 µL
- Alpha Tubulin:
- Use 3 aliquots of 4 µL each
- Add 200 µL PBS-Tw-Az-BSA to get 2X strength
- total volume = 600 µL
- Mix 1:1, and aliquot ~104 µL per pair. Make 10 aliquots
To mix secondaries
- Green anti-mouse:
- 2 1µL aliquot
- 300 µL PBS-Tw-Az-BSA
- total volume: 602 µL
- Red anti-rat:
- 2 5 µL aliquots
- 260 µL PBS-Tw-Az-BSA each
- total volume: 530 µL
- Mix 1:1, and aliquot ~106 µL per pair. Make 10 aliquots
10/03/23
10/03/23 kdorfman Mon, 10/02/2023 - 20:54Live Cell Staining & Imaging
Students use the dish they seeded previous Thursday.
Need "staining solution":
- Fluorobrite
- Nuc Blue
- Mitotracker Red
10/10/23
10/10/23 kdorfman Mon, 10/02/2023 - 19:38Kate transfects HCT116 cells with Eb1 plasmids.
Need 8-10 viewing dishes, seeded with transfected cells 1 to 2 days before viewing.
1 reaction needs ~ 106 cells/mL
100 uL cells per reaction (barely 8 drops)
So do 2 reactions, each with 106 cells.
2 days before, seed a 25 cm2 flask with 0.5 106cells.
(4 days before, seed with 0.125 106 cells)
1 drop of the 100 uL reaction into its own dish.
On transfection day, divide into 2 reactions
Notes on actual protocol followed:
Friday 10/6
- Start with 2 confluent 12.5 cm2 flasks
- (~1.25 x 106 cells)
- Need 1-5 x 106 cells/mL on Monday
- Split 1/8 to allow for 3 doublings
- Stop reaction with 1.6 mL
- Seed 0.2 mL into each of 2 new flasks.
Monday 10/9/23
- Put all the cells from one confluent 12.5 cm2 flask
- should be confluent again
- into a new 25 cm2 flask
Tuesday 10/10/23
- There should be 2.5 x 106 cells
- Trypsinize
- Stop reaction with 2 mL
- split suspended cells into two microfuge tubes.
- Spin down, and proceed with 2 Mirus nucleofection reactions
- Make 2 12.5 cm2 flasks
10/17/23
10/17/23 kdorfman Mon, 10/16/2023 - 19:26Trial run for Lamin antibodies
Fix 15 min in paraglut
1o Ab: Rabbit anti-lamin B
- dilute 1:1000 with PBS-Tw-Az-BSA
- 2 µL antibody into 2 mL buffer
- 1 hr 37C
2o Ab: goat anti-rabbit
- 1 hr RT incubation
Cell type | # coverslips |
---|---|
MEF WT NLS-GFP (40) | 3 |
HCT-116 WT (60) | 3 |
DU-145 (74) | 4 |
Stain with Hoechst (1:10,000) in the final PBS-Tw-Az rinse
Mount with non-DAPI Fluoromount (Fisher OB100-01)
10/24/23
10/24/23 kdorfman Mon, 10/16/2023 - 19:36Students begin projects
Kate provides fixed coverslips (seeded 10/22, fixed 10/24/23)
Cell line | medium | variant | treatment | no. coverslips |
---|---|---|---|---|
HCT116 | McCoy | WT (60) | 4 | |
LB1-AID-GFP (68) | 2 | |||
MEF | DMEM | WT (39) | control | 6 |
VPA | ||||
DZNP | ||||
LMNB1-/- (02) | 3 | |||
DMEM+G418 | LA KD (37) | 3 | ||
Prostate cancer | RPMI-1640 | DU145 (74) | 2 +3 from Mai | |
RPMI-1640 | LNCaP (77) | 2 + 3 from Mai | ||
DMEM | PC3 (76) | 2 |
Materials:
- Paraglut fix (enough for 30 wells)
- PBS to rinse 30 coverslips
- PB-TW-Azide (enough for 24 dunking beakers and 30 wells)
- 1o antibodies
- mouse anti-NP (nuclear pore)
- mouse anti-LB1
- rabbit anti-LB2 ab151735 (get one 10 µL aliquot from Drew - dilute 1/1000)
- 2o antibodies
- goat anti-mouse green for NP
- goat anti-rabbit red for LB1 & LB2
- Slides
- slide labels
- DAPI & droppers
- forceps
- dunking beakers
- humid chamber materials
Project | people | scope | IF coverslips | IF details |
---|---|---|---|---|
MEF VPA | Olivia & Juliana | 1 | 1x 39 unt, 1X 39 VPA, another VPA if pos | standard NP LB1 IF |
MEF DZNep | Karan & Nate | 2 | 1x 39 unt, 1X 39 DZNep, another if pos | standard NP LB1 IF |
MEF LMNB1-/- | Cole & Grif | 3 | 1X 39, 2X 02 LMNB1-/- | 39 & 02 NP LB2 IF, 1x 02 NP LB1 IF |
MEF LA KD | Shrushti, Emma, Anna | 8 | 1X 39, 2X 39 | standard NP LB1 IF |
HCT116 LB1 live 1 | Sam & Jillian | 5 | 68, 1x unt & 1x VPA | LB2 IF only |
HCT116 LB1 live 2 | Isabelle & Jason | 6 | 60, 1x unt 1x VPA; 60, 1x unt 1x VPA | one set NP LB1 IF; other set NP LB2 IF |
Prostate Cancer 1 | Tyian & Antonia | 4 | 2x 74, 1x 76 | standard NP LB1 IF |
Prostate Cancer 2 | Kelsey & Pedro | 7 | 2x 74, 1x 77 | standard NP LB1 IF |
2024 Bioimaging (Stephens)
2024 Bioimaging (Stephens) kdorfman Mon, 08/12/2024 - 22:37HeLas for teaching microscopy
MEFs for CURE
day | date | prep |
---|---|---|
Tu | 9/3 | 12 coverslips: HeLa (microtubules, actin, DAPI) |
Th | 9/5 | (Transmitted light and alignment) |
Tu | 9/10 | (Cameras, SNR, calibration) |
Th | 9/12 | (NA and resolution) beads |
Tu | 9/17 | (Fluorescence) |
Th | 9/19 | photobleaching (need new slides?) |
Tu | 9/24 | IF: need fixed coverslips, 10, 20 Ab |
Th | 9/26 | view IF from previous class, cell culture (MEF 39) |
Tu | 10/1 | Live cell imaging and cell culture |
Th | 10/3 | Plasmid prep Eb1 into MEF 39 and cell culture Kate transfects, puts into live dishes |
Tu | 10/8 | Transfection imaging |
Th | 10/10 | midterm |
Tu | 10/15 | NO CLASS (Monday schedule) |
Th | 10/17 | Begin CURE |
Tu | 10/22 | |
Th | 10/24 | |
Tu | 10/29 | |
Th | 10/31 | |
Tu | 11/5 | NO CLASS (Election Day) |
Th | 11/7 | |
Tu | 11/12 | |
Th | 11/14 | |
Tu | 11/19 | |
Th | 11/21 | |
Tu | 11/26 | CURE written report due |
Th | 12/3 | (Michael) Neurobiology |
Tu | 12/5 | (Michael) Neurobiology |
Tu | 12/10 | Flexible last day of class |
HeLa for 1st day
HeLa for 1st day kdorfman Wed, 08/28/2024 - 20:33For "Learn your Microscope" 9/3
9 HeLa (Vikash) coverslips
Fixed in paraglut
Stained for alpha tubulin:
Stained for green actin
Mounted in DAPI
Note: Red tubulin was gorgeous! Let's see if it lasts longer than the FITC.
IF for 9/24
IF for 9/24 kdorfman Wed, 08/28/2024 - 20:47Fixed coverslips (MEF 39 from Drew's lab)
Fix in paraformaldehyde
primary | secondary |
---|---|
Lamin B1 (rabbit, 1:1,000) | green |
NPC (mouse, 1:2,000) | red |
Emerin (mouse, 1:1,000) (from Drew's lab) | red |
To make the double primaries:
Make 2x Lamin B1 (add 1 mL instead of 2 mL)
Make 2x NPC (follow label directions = 2x (should say 1:20)
Make 2x Emerin: 1:500
Mix 250 µL each
To make the double secondary:
- Make 2x goat anti rabbit green
- goat anti rabbit green aliquots need 1194 µL to be 1X
- make 2X by adding 597 µL PBS-Tw-Az BSA
- Make 2X goat anti mouse red:
- aliquot says add 500 µL to make 1X
- add 250 µL to make 2X (use 2 aliquots)
- Make 1x green + red by mixing
- 500 µL goat anti rabbit green with
- 500 µL goat anti mouse red
DAPI
Slides
Beakers
forceps
Humid chambers
2024 Wadsworth
2024 Wadsworth kdorfman Tue, 01/16/2024 - 21:39UNIT 1
Lab | Day | date | materials |
---|---|---|---|
1 | Th | 2/1 | 1 slide each scope - DAPI |
2 | Tu | 2/6 | Same slide as Lab 1, micrometer |
3 | Th | 2/8 | 1 slide each scope, DAPI + 1 |
4 | Tu | 2/13 | Slides with DAPI, tubulin, actin, also beads for microscope resolution |
5 | Th | 2/15 | camera resolution; slides from Lab 4 |
6 | Tu | 2/20 | photobleaching. Fresh 3-color slide |
Th | 2/22 | NO CLASS - MONDAY SCHEDULE | |
Tu | 2/27 | Presentations |
UNIT 2
Lab | Day | date | materials |
---|---|---|---|
1 | Th | 2/29 | Immunofluorescence |
paraglut fixed coverslips1 for tubulin, phalloidin (2 each) | |||
materials for mounting, slide boxes | |||
live cells on coverslip for paraglut fix | |||
2 | Tu | 3/5 | live cells (GFP-tubulin LL's) in MatTek dishes mitotracker red ER tracker red NBD ceramide |
3 | Th | 3/7 | miscellaneous. grow some GFP E. coli |
4 | Tu | 3/12 | learn cell culture: 8 12.5 cm2 flasks of HeLa parentals |
catch-up day: | |||
12 dishes live LL-GFP-tub for ER tracker | |||
2 fixed coverslips for phalloidin staining | |||
student made slides from previous lab | |||
previously made instructor slides |
UNIT 3
Lab | Day | date | materials |
---|---|---|---|
1 | Tu | 3/26 | 9 coverslips HeLa, 6 coverslips HeLa treated with STLC 5 uM 18 hours, stained for alpha (2ogoat-anti-rat FITC>) and gamma (2ogoat-anti-mouse TRITC) tubulin |
2 | Th | 3/28 | HeLa: 3 coverslips, 1 flask, 3 dishes |
LLCPk-1 GFP alpha 10-15 dishes | |||
taxol | |||
nocodazole | |||
GSK923295 (cenp inhibitor) | |||
cyto-D | |||
RO3306 (CDK-1 inhibitor) | |||
3 | Tu | 4/2 | HeLa coverslips; LLCPk alpha GFP dishes |
4 | Th | 4/4 | HeLa: 2 live, 2 coverslip; LLCPk alpha GFP: 10 live, 4 coverslip |
5 | Tu | 4/9 | HeLa: 3 live, 5 coverslips; LLCPk-1: 10 live, 3 coverslips |
6 | Th | 4/11 | HeLa: 5 coverslips, 2 dishes; LLCPk-1: 10 live |
7 | Tu. | 4/16 | HeLa: 4 coverslips; LLCPk-1: 10 live |
Unit 4
For first day Tu, 4/23: 12 HeLa coverslips
- fixed in methanol
- stained with rat anti alpha tubulin, goat-anti-rat FITC), and 3 stained with each of:
antibody | final dilution |
---|---|
rabbit anti Kif2a | 1:1000 |
rabbit anti-CAMSAP-2 | 1:500 |
rabbit anti-CAMSAP-1,2,3 | 1:100 |
mouse anti-gamma tubulin | 1:20 |
with goat anti-rabbit red as the secondary
Plasmids for Unit 4 from Addgene
Gene | Addgene # | bacterial selection | mammalian selection |
---|---|---|---|
Kif2a-EGFP | 52401 | Kan | Neomycin (G418) |
CAMSAP-3 neon green | 191322 | amp | |
EGFP-alpha tubulin | 12298 | amp | Neomycin (G418)/Kan |
Mcherry tubulin | 31930 | Kan | Neomycin (G418) |
mCherry H2b | 20972 | Kan (amp) | Neomycin (G418) |
Project 2
Project | grp | gene | Thu 4/25 | Tu 4/30 | W 5/1 | Th 5/2 |
---|---|---|---|---|---|---|
Stable cell line | 1 | Kif2A | mini prep & Nanodrop (Tom) | nucleofect need 100K cells/flask | observe dish, select flask | |
6 | Kif2A | |||||
8 | Camsap 3 | |||||
RNAi | 2 | Camsap2 | HeLa live (KD), HeLa mCherry (Pat) | 6 well plates (200 - 250K cells) | coverslips | Fix & Stain |
3 | spastin, katanin | |||||
4 | camsap3 | |||||
5 | spastin | |||||
7 | Kif2A |
-
HeLa or LLCPk1 ↩︎
Cell culture
Cell culture kdorfman Mon, 07/09/2012 - 19:38Tissue Culture Facility
Tissue Culture Facility margaret Thu, 10/27/2011 - 20:27Hoods When beginning work in a hood, spray all walls and the working surface with 70% ethanol and wipe clean.
When you have finished working in a hood: please leave it empty, spray the walls and working surface with 70% ethanol and wipe, and make sure that the gas is off. Leave the fan on and turn on the UV light.
Incubators Immediately remove any contaminated dish or flask from the incubator and treat itwith 30% bleach. Wipe the shelf it was on with 70% ethanol. Notify any individuals with cells in the incubator of the contamination.
If you are using an incubator that requires CO2: Always check the water level in the bottom of the incubator, if level is low add water. You are responsible for checking the level of CO2 in the tanks, if pressure falls below 800 psi the tank is emptying. After having received instructions, connect a full tank to the incubator and order a replacement (or notify someone).
Microscopes Please make sure the microscopes are clean and covered when not in use.
Pipets Dispose of all glass Pasteur pipets in the glass only bin. Disposable plastic pipets should be placed in trash (in the plastic sleeve). Reusable pipets should be placed tip up in the pipet jar.
Supplies Label all items in the refrigerator with your name and date. Items that not labeled will be discarded.
Vacuum Pump When you are done, please empty and rinse flask with 30% bleach.
Other Keep the area clean at all times. Dishes and flasks of cells to be discarded should be treated with 30% bleach before trashing.
Bulk coverslip staining
Bulk coverslip staining kdorfman Wed, 09/08/2021 - 20:03Grow cells on coverslips in 6-well plates
Remove coverslips using a fine-tipped forceps when desired cell density is reached, and put in coverslip rack, cell side facing "cells" label on holder.
![](coverslip rack.jpg)
Put holder in 100 mL beaker containing ~60 mL PBS with a tiny stir bar. Rinse by stirring for ~5 minutes.
Transfer holder to a second 100 mL beaker containing ~60 mL of fixative. Fix with stirring for ~10 minutes
Transfer holder to a 3rd beaker containing PBS-Tw-Az. Rinse by stirring for ~5 minutes.
Repeat with another beaker of PBS-Tw-Az.
Stain or mount coverslips right away, or store them in the holder in a brown jar with fresh PBS-Tw-Az. They may last up to a month in the refrigerator. Longer than that, they will start to fall off the coverslip.
CO2 Incubator
CO2 Incubator kdorfman Mon, 07/09/2012 - 19:41Thermo Scientific Napco Series 8000 DH
Room | Model | Serial Number |
---|---|---|
368 | 3584 | 310670-97 |
371 | 3598 | 313043-1035 |
1-888-213-1790
- 37C
- 5% CO2
- pan of water in bottom
Thermo Scientific Isotemp Gas Line Filter:
- Fisher 15-497-026 (Thermo Scientific 760210) $176.54/Pack of 10
Thermo Scientific Replacement HEPA Main Filter:
- Fisher 15-497-022 (Thermo Scientific 760175) $60.60 each
- Fisher 15-497-023 (Thermo Scientific 760209) $308.62/Pack of 4
CO2 sensor for model 3598: part # 1900602 (~$400)
Manuals under incubator in 371
Counting cells
Counting cells kdorfman Thu, 09/12/2019 - 16:58Just before plating, after cold medium has been added to the trypsinized cells,
mix 0.1 mL cell suspension with 0.1 mL 0.4% trypan blue. Live cells will not take up trypan blue. (Use a different dilution factor if cells are uncountable)
inject 10 uL to one side of the hemocytometer (see image attached below).
Inspect at 10x.
Count the number of live (clear) cells in all 9 squares of the hemocyometer. (Volume = 3mm * 3 mm * 0.1 mm = 0.9 uL) (Count in 4 1mm x 1mm squares if cells are very numerous)
Calculate the live cell concentration (# live cells * 1.11)/1uL for 9 squares,
(# live cells * 2.5/uL for 4 squares)correct for dilution factor
Estimating cell numbers
Estimating cell numbers kdorfman Tue, 08/22/2023 - 17:23From Useful Numbers for Cell culture
(See also counting cells)
Container | Surface area (cm2) | Seeding density* | Cells at confluency | mL growth medium |
---|---|---|---|---|
35 mm dish | 8.8 | 0.3 x 106 | 1.2 x 106 | 2 |
60 mm dish | 21.5 | 0.8 x 106 | 3.2 x 106 | 5 |
100 mm dish | 56.7 | 2.2 x 106 | 8.8 x 106 | 12 |
150 mm dish | 145 | 5.0 x 106 | 20.0 x 106 | 30 |
6-well plate | 9.6 | 0.3 x 106 | 1.2 x 106 | 1 to 3 |
12-well plate (~20 mm) | 3.5 | 0.1 x 106 | 0.5 x 106 | 1 to 2 |
24-well plate (~15 mm) | 1.9 | 0.05 x 106 | 0.24 x 106 | 0.5 to 1.0 |
48-well plate | 1.1 | 0.03 x 106 | 0.12 x 106 | 0.2 to 0.4 |
96-well plate | 0.32 | 0.01 106 | 0.04 x 106 | 0.1 to 0.2 |
T-12.5 flask | 12.5 | 0.35 x 106 | 1.4 x 106 | 1.5-2.5 |
T-25 flask | 25 | 0.7 x 106 | 2.8 x 106 | 3–5 |
T-75 flask | 75 | 2.1 x 106 | 8.4 x 106 | 8–15 |
T-175 flask | 175 | 4.9 x 106 | 23.3 x 106 | 35–53 |
T-225 flask | 225 | 6.3 x 106 | 30 x 106 | 45–68 |
- Seeding density is given for each culture vessel type as follows:
- Dishes and Flasks: Cells per vessel;
- Culture plates: Cells per well
Hemocytometer Cautions
Hemocytometer Cautions kdorfman Fri, 05/29/2020 - 13:11Use of the Hemacytometer for the Determination of Cell Numbers
Counting cells by the use of a hemacytometer is a convenient and practical method of determining cell numbers in the case that the Coulter counter is out-of-order temporarily. (It is not that bad.) The hemacytometer consists of two chambers, each of which is divided into nine 1.0 mm squares. A cover glass is supported 0.1 mm over these squares so that the total volume over each square is 1.0 mm x 0.1 mm or 0.1 mm3, or 10-4 cm3. Since 1 cm3 is approximately equivalent to 1 ml, the cell concentration per ml will be the average count per square x 104.
Hemacytometer counts are subject to the following sources of error:
- 1. Unequal cell distribution in the sample
- 2. Improper filling of chambers (too much or too little)
- 3. Failure to adopt a convention for counting cells in contact with the boundaries lines or with each other (be consistent)
- 4. Statistical error
With careful attention to detail, the overall error can be reduced to about 15%. It is assumed that the total volume in the chamber represents a random sample. This will not be a valid assumption unless the suspension consists of individual well-separated cells. Cell distribution in the hemacytometer chamber depends on the particle number, not particle mass. Thus, cell clumps will distribute in the same way as single cells and can distort the result. Unless 90% or more of the cells are free from contact with other cells, the count should be repeated with a new sample. A sample will not be representative if the cells are allowed to settle before a sample is taken. Always mix the cell suspension thoroughly before sampling. The cell suspension should be diluted so that each such square has between 20 - 50 cells (2-5 x 10 5 cells/ml). A total of 300 - 400 cells should be counted, since the counting error is approximated by the square root of the total count. A common convention is to count cells that touch the middle lines (of the triple lines) to the left and top of the square, but do not count cells similarly located to the right and bottom. Hemacytometer counts do not distinguish between living and dead cells. A number of stains are useful to make this distinction. Trypan blue among others (Erythrosin B, Nigrosin) can be used: the nuclei of damaged or dead cells take up the stain. If more than 20% of the nuclei are stained, the result is probably significant. Although the trypan stain distinction has been questioned, it is simple and gives a good approximation. See procedure here
References:
From the Laboratory of Dr. Allan Bradley
Baylor College of Medicine, Houston, Texas
Culturing S-cells
Culturing S-cells kdorfman Fri, 05/29/2020 - 15:44Cells are in suspension -- they are not dead!
To make conditioned medium:
- spin cells to bottom of conical tube (be gentle!!).
- Save the supernatant in a labeled tube that has the date and is labeled “conditioned medium”.
- Store extra in fridge.
To grow cells:
- Grow cells at room temperature, no CO2.
Passage every 3-4 days. Dilute cells 1:5 each split and use 20% conditioned medium.
Put cells from flask in tube; spin; take off and save supernatant (this is your conditioned medium!)
- Resuspend pellet (gently) in 5 mL medium.
- Add to clean flask
- 1 mL conditioned medium (supernatant)
- 1 mL cell suspension
- 3 mL fresh medium
- To induce gene expression (e.g, tubulin) add copper sulfate to the medium
- cells are neomycin resistant
- Induce 1 day prior to imaging
- to image put on previously prepared ConA coated coverslips. Image 30 min after plating on Con-A coated dishes or Con-A coated coverslips
To make medium for growth of Drosphila cells:
Schneider's medium + 10% heat-inactivated FCS.
Freezing S2 cells
Spin down a 3-4 day old 75 cm2 confluent flask. 1200 RPM, 5 min
Remove conditioned medium
Resuspend one flask's worth of cell pellet in "freezing medium"
- 1.8 mL fresh medium (including antibiotic and serum)
- 1.8 conditioned
- 400 uL DMSO
Make 1mL aliquots.
Put in -80 in styrofoam for 1 day.
Transfer to liquid nitrogen.
Freezing
Freezing kdorfman Mon, 07/09/2012 - 19:59Approximately 1 freezing vial per 25 cm2 flask.
3 vials from large size (75 cm2) culture flask.
Feeding Cells before freezing
- When cells are 90% confluent (~4 days), replace medium
- Draw out old medium, replace with 5 mL new (warm) medium.
Freezing cells
- Make freezing medium (15% DMSO, 20% serum) in advance
- Remove medium from flask
- Rinse once with ~3 mL sterile PBS. (Squirt in, then suck out.)
- Add 10 – 15 drops trypsin (0.5 – 0.75 mL)
- Incubate (37C) 5 – 30 min
- Add 3 mL cold medium (regular)
- Pour into 15 mL centrifuge tube
- Optional – rinse with another 2 mL, to capture as many cells as possible
- Spin (~100-500xg) 5 – 10 min (until supernate is clear and pellet is hard)
- Pour off supernate (if pellet is solid – otherwise pipette it off)
- Resuspend in 1 mL freezing medium (pipette up and down)
- Transfer to freezing vial (should be slightly more than 1 mL)
- Put on ice
- Store overnight in -80C in "Mr. Frosty" (actually a VWR 414004-284 CryoCooler Freeze Controller)
- Put into liquid nitrogen.
Fill out the freezing log
Nucleofection
Nucleofection kdorfman Tue, 09/25/2012 - 18:08Mirus
Mirus kdorfman Fri, 12/06/2019 - 14:36general summary
100 uL Mirus reagent
2 ug DNA(up to 20 uL DNA solution)
1 - 5 million cells/mL
Lonza Knowledge Center
Protocol
- Warm medium in flask and coverslip-bottom dish
- Mix DNA and Mirus reagent; warm up
- Trypsinize cells:
- remove medium
- rinse with warm PBS
- add 0.5 mL trypsin
- incubate till all cells are in suspension (at least 3 min)
- add 1 mL COLD medium
- mix well by pipetting up and down
- transfer all to sterile 2 mL tube
- spin 500 x g 3 minutes
- resuspend cells in Mirus/DNA solution
- put all into sterile cuvette, close
- Put cuvette into Nucleofector
- Choose program1
- Press the button
- Use special dropper to transfer one drop to the coverslip dish and the rest to the waiting flask.
-
Cell type Program # B16 P-031 3t3 U-030 HCT116 D-032 HeLa I-013 LLCPk-1 X-001 MEF T-020, A-023
Cell Density
Cell Density kdorfman Wed, 09/29/2021 - 16:47selection of nucleofected cells
selection of nucleofected cells kdorfman Thu, 10/03/2019 - 20:47Addgene plasmid selection
Addgene plasmid selection kdorfman Sat, 05/30/2020 - 13:52When cells are medium-light, replace medium with 0.5 g/L G418 (= 0.5 mg/mL)
Next day, replace with medium with 1 g/L (=1 mg/mL)
Next day, 2 g/L (=2 mg/mL)
Solution is 50 mg/mL
Dilute 10 uL per mL to get 0.5 mg/mL
Dilute 20 uL per mL to get 1 mg/mL
Dilute 40 uL/mL to get 2 mg/mL
Making a Stable Cell Line Expressing Your Favorite Gene
Making a Stable Cell Line Expressing Your Favorite Gene margaret Fri, 10/21/2011 - 14:38- Make a death curve. This is to determine the minimal concentration of antibiotic that kills cells that do not have the resistance gene.
- transfect the cells with the plasmid containing the gene that you want to express in the cells (see Amaxa protocol).
- incubate the cells for about 48 hours after transfection so that the gene is expressed, and the resistence gene is expressed.
- add the appropriate dose of antibiotic (this is the selection step). The cells that do not contain the resistance gene (i.e. that were not successfully transfected) will die. Change the medium every 2 or 3 days to remove dead cells. Replace medium with fresh medium containing the antibiotic.
- split the cells as needed. Use medium with antibiotic. Make some coverslips to see if the cells are expressing the gene of interest.
After ~2 weeks any cell without the transgene and selectable marker should have died. The remaining cells are a mixed population – different cells might express different levels of the transgene. These can be used for preliminary experiments.
Making a clonal cell line
- 1. prepare cloning rings by smearing a small amount of grease on one end; make about 10. Have sterile forceps ready; have trypsin ready. Have a 24 well plate ready with some medium in the number of wells that you are planning to use.
- 2. remove medium from dish.
- 3. rinse with PBS--; leave a little bit so the cells don't die.
- 4. tilt the dish to see the colonies; place a cloning ring over nice large and well separated colonies. Press down firmly. Don't let the ring slide around. Add ~100ul of trypsin to each ring; wait a few mins. Pipette cells up and down (in the ring) add the released cells to one well of a 24 well plate. Each well now has a colony of cells – a potential cell line. When they grow, split each well – place some cells in a well of a six well plate to grow some more and put some in a Matek dish – be sure to label carefully so you know which is which. When the cells in the Matek dish are grown – check them in the fluorescence microscope. If they all have the same level of fluorescence and are healthy looking, they are a potential line (which you can further characterize with a Western Blot to quantify the level of expression).
Toss any cell lines that are not healthy or have too much or too little fluorescence.
Lonza Amaxa
Lonza Amaxa kdorfman Fri, 12/06/2019 - 14:34Nucleofection with Lonza Amaxa
Order VCA-1005 Cell Line Nucleofector Kit L (25 reactions $347)
Transfection Protocol
- Warm medium in MatTek dishes to equilibrate CO2
- Warm additional 0.5 mL medium in sterile microfuge tube
- Make mucleofection solution, RT, per flask:
- 19 µL supplement 1
- 81 µL reagent L for LLCPk (Kit R for 3t3, HeLa)
- Remove medium from flask
- Rinse with PBS
- Add trypsin
- Incubate ~5 min, or until cells are floating
- Add cold medium
- Transfer to sterile 15 mL centrifuge tube
- Spin 10 min, slow speed
- Remove medium. Pellet should be soft
- Loosen pellet by flicking tube
- Add all 100 µL nucleofection solution plus the DNA (one of the following):
- 2 µg plasmid DNA
- 5 µg BAC
- 5 µL siRNA (20 µM)
- Pipet up and down to mix
- Transfer to nucleofection cuvette
- Insert into nucleofector device, choose program
- X001 for LLCPK
- I-013 for HeLa
- U-030 for 3t3
- Resuspend cells in 500 µL warm medium
- Distribute in drops on coverslips of MatTek dishes
Plating
Plating kdorfman Mon, 07/09/2012 - 19:50- Put medium into dishes
- MatTek dishes for observations of live cells
- coverslip in a 35 mm dish for fixation and mounting
- Let dishes equilibrate in incubator
- Treat cells in flask with trypsin as for splitting
- Stop trypsinization with cold medium
- Add 1 - 4 drops of medium + cells to each dish, depending on cell density
- Check density of first dish, adjust volume for the remaining dishes
- Check after 24 hours.
Splitting
Splitting kdorfman Mon, 07/09/2012 - 19:44- Put 5 mL medium in each flask. 2.5 ml for mini flasks. Label with cell type and passage number
- Warm flasks in incubator
- Remove medium from cells.
- Right away, Rinse once with ~3 mL sterile PBS. (Squirt gently in, Rock the flask, and then suck out. Do not hit the place where cells are. )
- Add 15 drops sterile trypsin (=~0.75 mL) - enough to cover cells, if more than a thin layer, pull some out
- Incubate (37C) ~5 min or until all floating, likely no more than 5m.
- Check to make sure you can see them floating in the liquid
- Add ~1mL medium to flask, draw up and down to mix.1
- Put back in incubator.
-
Use a ratio of medium: trypsin of 2:1 if you need the cells to hang out longer (say for counting) before going into the new flask. ↩︎
-
if original flask was totally confluent, then use ~1/10th to ~1/5th of the volume; use more if you need cells in a day or two, use less if you don't need cells for a few days. ↩︎
-
For dishes: if you want a nice monolayer of cells to form within one or two days, add a drop or two of cells AND examine in the microscope. You can quickly get a feel for the number of rounded cells and how heavy the plating will be. You can measure the exact volume and keep track if that helps. Remember to rock to distribute cells on the coverslip or Mattek surface (glass bottom dish). Do not swirl. ↩︎
Thawing
Thawing kdorfman Mon, 07/09/2012 - 19:40- Label flask with cell line, passage number, date
- Put 10 mL medium in flask, set in incubator
- Retrieve frozen vial from LN2 dewar, thaw in incubator or water bath.
- Put ~1 mL thawed cells into a single flask
- 24 hours later, replace medium (5 mL)
- Split as needed
Labs 2012 & before
Labs 2012 & before kdorfman Thu, 09/26/2013 - 14:511.1: intro to microscopes
1.1: intro to microscopes kdorfman Tue, 12/27/2011 - 21:091.2: image formation
1.2: image formation kdorfman Tue, 12/27/2011 - 21:102.1: Numerical aperture
2.1: Numerical aperture kdorfman Tue, 12/27/2011 - 21:10need dapi slides - can use the same ones for 2.2
squuze bottle of ethanol to clean oil off lenses.
Stage micrometer:
10X: 2.162 pixels/µm
40X: 8.728 pixels/µm
Reticle is 2 mm
Major divisions are 100 µm
tiniest divisions are 10 µm
2.2: Resolution
2.2: Resolution kdorfman Tue, 12/27/2011 - 21:113.1: Fluorescence
3.1: Fluorescence kdorfman Tue, 12/06/2011 - 15:33Fluorescence Microscopy
LLCPK fixed and stained with DAPI.
Prepare before the semester starts, keep in a slide box in the refrigerator.
Bring to room temperature in the morning of the lab to minimize condensation on coverslip.
Keep track of which number slides have been used, so they will not be used later in any lab where photobleaching is an issue.
3.2: Immunofluor
3.2: Immunofluor kdorfman Wed, 11/23/2011 - 16:55Immunofluorescence Labeling
Turn on the Incubator
Reagents
PBS (warm) for rinsing medium off cells before fixation
PBS-Tw-Az for rinsing coverslips (in carboy)
2% BSA in PBS to make antibodies
Fixative
- paraglut for students
- methanol in freezer for certain antibodies
Slides
Nail polish
Mystery Antibodies for 2012
No. | Antibody | from | fixative | secondary | filter cube |
---|---|---|---|---|---|
1 | Golgi | KD | formaldehyde | goat anti mouse | G |
2 | LAP2 | PW | methanol | goat anti mouse | G |
3 | gamma tub | PW | paraglut or form | goat anti rabbit | G |
4 | ZO1 | KD | methanol or paraform | goat anti rabbit | G |
5 | vinculin | PW | formaldehyde | goat anti mouse | G |
Cells for 3.2
Cells for 3.2 kdorfman Tue, 12/06/2011 - 15:01Immunofluorescence Labeling
3.3: Direct fluor
3.3: Direct fluor kdorfman Tue, 12/06/2011 - 15:57Direct Fluorescent Labeling of Organelles
CHECK THIS FOR HBS vs PBS!!
Cells
3t3, LLCPk
- Fixed in paraglut for phalloidin
- 8 3t3 (can be fixed as late as the morning of lab)
- 8 LLCPk (can be from pre-semester prep)
- Live on small diameter MatTek dishes for Mitotracker and ceramide
- 8 3t3
- 8 LLCPk
Reagents
HBS to incubate ceramide
3.4 GFP-tags
3.4 GFP-tags kdorfman Tue, 09/25/2012 - 17:47Live cells on MatTek dishes:
Task 2. GFP-alpha-tubulin cells (everybody) 8 dishes
Task 3. Nucleofected with GFP version of (2 different ones per group) 4 - 8 dishes each:
- Rab11
- E cadherin
- H2B (GFP)
- alpha actinin
Lipofectamine
Lipofectamine kdorfman Tue, 09/25/2012 - 17:56Lipofectamine2000 Plasmid Transfection
24-48 hrs before class,
- transfect cells (at 40-60% confluency)
- Medium without
- antibiotic
- serum
- L-glutamine
Protocol
- Dilute DNA with DMEM
- Dilute lipofectamine2000 with DMEM, incubate <5 min at RT
- Mix DNA with Lipofectamine2000, incubate >15 min at RT
- Rinse 2x with DMEM
- Add complexes to cells, incubate 4 hr 37C
- Rinse 2x with warm PBS
Add normal medium back to cells
Per 35 mm dish (1 coverslip):
- 5 µg DNA in 500 µL
- 10 µL lipofectamine in 500 µL
Plasmids
Plasmids kdorfman Tue, 09/25/2012 - 21:03Addgene plasmids have G418 (neomycin) resistance except 26737
(E. coli for plasmids are in -80)
gene | fluorophore | resistance | mg/mL (Abs 280) | mg/mL (260/280) | µL to get 2 µg | source |
---|---|---|---|---|---|---|
rab11 WT | GFP | kan | 6.3 | 7.8 | 0.6 | Addgene 12674 |
E cadherin | EGFP | amp | 2.5 | 2.5 | 1 | PW |
H2B | m cherry | kan | 1.8 | 1.3 | PW | |
H2B | EGFP | kan | 5 | 7.3 | 0.33 | PW |
EB1 | EGFP | kan | 0.662 | 0.86 | too low | PW |
Actin | EGFP | kan | 5.2 | 4.97 | 0.4 | PW |
a-actinin | EGFP | kan | 1.29 | 2.73 | 1 | PW |
life act | GFP | kan (G418 for transfected selection - 1mg/mL for 3t3) | 99.1 ng/uL | 280: 1.054 | 260: 1.983 | Addgene 58470 |
H2B | mCherry | Amp | Addgene 20972 | |||
UtrCH | GFP | Amp | Addgene 26737 | |||
E-cadherin | GFP | Amp | Addgene 28009 | |||
Sec61 beta | mcherry | Kan | Addgene 49155 | |||
Lifeact 7 | mcherry | Kan | Addgene 54491 | |||
Golgi 7 | mcherry | Kan | Addgene 55052 | |||
LaminA-C-18 | mcherry | Kan | Addgene 55068 | |||
Lysosomes-20 | mcherry | Kan | Addgene 55073 | |||
Mito-7 | DsRed2 | Kan | Addgene 55838 | |||
Vimentin-7 | EGFP | Kan | Addgene 56439 | |||
Sec61b-C1 | mEmerald | Kan | Addgene 90992 |
Making plasmids
PrepEase Endotoxin-Free Maxi Plasmid Kit, 78730 from USB
Streak cells on LB-30 µg/mL Kan or LB-amp 50 µg/mL (?)
Pick a single colony.
Inoculate 100 mL LB (+ 50 – 100 µg/mL ampicilin or 10 – 50 µg/mL kanamycin)
Shake overnight, 37C
Divide entire culture into 3 or 4 50-mL conicals
Use the appropriate rotor in the biochemistry floor model centrifuge.
Follow PrepEase directions, except: let filtrate drip directly into column (Steps 5-6)
Test in spectrophotometer at 1:500. Adjust dilution as needed
- Abs 260
- 260/280 ratio
DNA concentration
DNA concentration kdorfman Fri, 10/01/2021 - 19:224.1: Cell cycle
4.1: Cell cycle kdorfman Mon, 11/14/2011 - 21:58Cell Cycle
Fixed and mounted coverslips of LLCPk cells, stained with DAPI and anti-tubulin
normal
serum starved
Cells won't adhere to the coverslip without serum, so grow them first for 24 hours in regular medium.
Remove the medium, replace with serum-free medium
Fix the coverslips 24 hours later.
- Nocodazole treated
5.1: Motility
5.1: Motility kdorfman Mon, 11/14/2011 - 21:57Motility
Live cells on MatTek dishes
8 MatTek dishes each:
3t3
B16 (do more than one set, at different concentrations)
LLCPK actin
Must be plated lightly for motility.
Scheduling
Thaw LLCPK-actin, B16 24 hours before plating
Plate on all cells (lightly) Monday for Wednesday lab
3t3’s must have 36-48 hours to stretch out.
If they get overgrown (especially B16s), scrape some off
Reagents
Aliquot non-CO2, serum-free medium
5.2: Motility Projects
5.2: Motility Projects kdorfman Wed, 10/12/2011 - 02:36CHECK CYTOCHALASIN STOCKS FOR 2012
2012: 500 nM (250 nM final in dish) should give an effect without causing massive detachment of cells
Stock is 10 mM in DMSO.
Need 6 1 mL aliquots of 500 nM in non-CO2 medium:
- 1 µL 10 mM stock
- 20 mL medium
2011: working concentration of cytochalasin D: 1-10uM
Make 200uM stock for students to dilute with non-CO2 medium
4 groups using cytochalasin, each doing 4 dishes.
Maximum conc 10 µM, in 2 mL
vi = (10 µM * 2 mL)/200 µM = 0.1 mL
So each group would use at most 0.4mL
Stock was 10 µL of 10 mM cytochalasin D in DMSO.
Adding 450 µL of non-CO2 medium makes it 500 µL of 200 µM
cf = (10 mM * 10 µL)/500 µL = 0.2 mM = 200 µM
Frozen stock is 2.5 mM
Invitrogen PHZ1063
Make 500 µL of 0.2mM
vi = (0.2mM * 500 µL)/2.5 mM = 40 µL
Add 460 µL non-CO2 medium
40 µL aliquots. Each can make 500 µL of 200 µM for use.
Nocodazole
Taxol
6.1: Endocytosis
6.1: Endocytosis kdorfman Tue, 10/11/2011 - 22:33Receptor-mediated Endocytosis
Monday 10/17/11
Materials for 6.1
Materials for 6.1 kdorfman Thu, 10/13/2011 - 18:54- fine forceps to pick up coverslips
- humid chamber petri dishes + filter paper (4 per group)
- ice in ice bucket
- small beakers to rinse coverslips
- glass slides
- mounting medium
- nail polish
cells & reagents 2011
cells & reagents 2011 kdorfman Wed, 10/10/2012 - 21:07Cells on coverslips
Cells on coverslips kdorfman Tue, 10/11/2011 - 22:37Reagents for 6.1
Reagents for 6.1 kdorfman Tue, 10/11/2011 - 22:38Fe-citrate
If you used 10 mM Fe-citrate, instead of 89.4 mM, the calculations would be easier.
The 89.4 value is because Tobias Baskin's lab makes a 89.4 µM Fe-citrate medium, and it's easy to mix up a batch from the 1000x stock solution.
- Log in to post comments
For convenience, all staining, incubation, etc. will be done in HBS.
Each group does 4 coverslips, all of the same cell type. Some will use LLCPk, others will use 3t3.
HBS to mix everything else in. There is a bottle of sterile 10X on the prep room bench.
- 6 mL aliquots COLD (final rinse before fix coverslips 1-3)
- 15 mL aliquots RT in hood (to rinse off fixative)
- Need ~300 mL total
Fe-HBS for coverslips 1-3
- 20 mL aliquots COLD to rinse off growth medium, then tf
- 5 mL aliquots WARM (incubation after tf treatment)
- need some to make Fe-HBS-BSA for transferrin
- make 200 mL
Solution | stock conc | final conc | 25 | 50 | 75 | 100 | 200 | mL |
---|---|---|---|---|---|---|---|---|
10X HBS | 10 | 1 | 2.5 | 5 | 7.5 | 10 | 20 | mL |
Fe-citrate (mM) | 89.4 | 0.1 | 27.96 | 55.92 | 84 | 112 | 224 | µL |
plus water to final volume
Fe-HBS-BSA 1 mg/mL to make transferrin solution
- 5 mg BSA in 5 mL Fe-HBS
high-Fe-HBS for coverslip 4
- 2 mL aliquots COLD
- 2 mL aliquots WARM
- need some to make high-Fe-HBS-BSA for transferrin
- Make 50 mL
Solution | stock conc | final conc | 10 | 15 | 20 | 25 | 30 | 50 | mL |
---|---|---|---|---|---|---|---|---|---|
10X HBS | 10 | 1 | 1 | 1.5 | 2 | 2.5 | 3 | 5 | mL |
Fe-citrate (mM) | 89.4 | 2 | 223.7 | 335.6 | 448 | 559.2 | 671 | 1118.4 | µL |
plus water to final volume
High-Fe-HBS-BSA 1 mg/mL to make transferrin solution
- 5 mg BSA in 5 mL High-Fe-HBS
Transferrin in Fe-HBS-BSA to treat coverslips 1-3.
- Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM). Final concentration = 1 µL.
- Each group does 3 50-µL treatments (need extra for pipetting error)
- Make 1.2 mL: 19.2 µL AF-488-Tf + 1.1808 mL Fe-HBS-BSA
- Aliquot 165 µL
if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.
# aliquots | 3 | 6 | 9 | 12 | 15 | 18 | 21 | 24 | |
---|---|---|---|---|---|---|---|---|---|
vi (Tf) | 2.64 | 5.28 | 7.92 | 10.56 | 13.2 | 15.84 | 18.48 | 21.12 | µL |
HBS | 162.4 | 324.7 | 487.1 | 649.4 | 811.8 | 974.2 | 1136.5 | 1298.9 | µL |
vf | 165 | 330 | 495 | 660 | 825 | 990 | 1155 | 1320 | µL |
- Transferrin in high-Fe-HBS-BSA
- Each group does 1 50 µL treatment.
- Make 0.44 mL: 7 µL AF-488-Tf + 0.433 mL high-Fe-HBS-BSA
- Aliquot 55 µL
# aliquots | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
---|---|---|---|---|---|---|---|---|---|
vi (Tf) | 0.88 | 1.76 | 2.64 | 3.52 | 4.4 | 5.28 | 6.16 | 7.04 | µL |
HBS | 54.1 | 108.2 | 162.4 | 216.5 | 270.6 | 324.7 | 378.8 | 433.0 | µL |
vf | 55 | 110 | 165 | 220 | 275 | 330 | 385 | 440 | µL |
non-CO2 medium ICE COLD USE Fe-HBS - change manual !!!
- 3 rinses per coverslip
- 15 mL aliquots in the refrigerator
- need over 100 mL
HBS-paraformaldehyde 3.7%
- Need 60 mL
- Divided in 2 aliquots, one per fume hood
Ingredient units 10 15 20 25 40 50 60 paraformaldehyde g 0.37 .555 2 0.925 1.48 1.85 2.22 water mL 9 13.5 18 22.5 36 45 57.78 10X HBS mL 1 1.5 2 2.5 4 5 6
heat paraformaldehyde in water, then add 10X HBS
Add a drop of 10N NaOH
Heat and stir in the fume hood
cells & reagents 2012
cells & reagents 2012 kdorfman Wed, 10/10/2012 - 21:07Cells for 6.1
Cells for 6.1 kdorfman Wed, 10/10/2012 - 21:15Cells for 6.1
3t3 on coverslips
3 dishes per group, plus extras = 24
Plate 2 days before lab.
Reagents 2012
Reagents 2012 kdorfman Wed, 10/10/2012 - 21:16Omit the high-iron treatment
For convenience, all staining, incubation, etc. will be done in HBS.
Each group does 3 coverslips of 3t3.
HBS to mix everything else in. There is a bottle of sterile 10X on the prep room bench.
- 6 mL aliquots COLD (final rinse before fix coverslips 1-3)
- 15 mL aliquots RT in hood (to rinse off fixative)
- Need ~300 mL total
Fe-HBS
- 20 mL aliquots COLD to rinse off growth medium, then tf
- 5 mL aliquots WARM (incubation after tf treatment)
- need some to make Fe-HBS-BSA for transferrin
- make 200 mL
Solution | stock conc | final conc | 25 | 50 | 75 | 100 | 200 | mL |
---|---|---|---|---|---|---|---|---|
10X HBS | 10 | 1 | 2.5 | 5 | 7.5 | 10 | 20 | mL |
Fe-citrate (mM) | 89.4 | 0.1 | 27.96 | 55.92 | 84 | 112 | 224 | µL |
plus water to final volume
- Fe-HBS-BSA 1 mg/mL to make transferrin solution
- 5 mg BSA in 5 mL Fe-HBS
Solution | stock conc | final conc | 10 | 15 | 20 | 25 | 30 | 50 | mL |
---|---|---|---|---|---|---|---|---|---|
10X HBS | 10 | 1 | 1 | 1.5 | 2 | 2.5 | 3 | 5 | mL |
Fe-citrate (mM) | 89.4 | 2 | 223.7 | 335.6 | 448 | 559.2 | 671 | 1118.4 | µL |
plus water to final volume
- Transferrin in Fe-HBS-BSA
- Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM). Final concentration = 1 µL.
- Each group does 3 50-µL treatments (need extra for pipetting error)
- Make 1.2 mL: 19.2 µL AF-488-Tf + 1.1808 mL Fe-HBS-BSA
- Aliquot 165 µL
if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.
# aliquots | 3 | 6 | 9 | 12 | 15 | 18 | 21 | 24 | |
---|---|---|---|---|---|---|---|---|---|
vi (Tf) | 2.64 | 5.28 | 7.92 | 10.56 | 13.2 | 15.84 | 18.48 | 21.12 | µL |
HBS | 162.4 | 324.7 | 487.1 | 649.4 | 811.8 | 974.2 | 1136.5 | 1298.9 | µL |
vf | 165 | 330 | 495 | 660 | 825 | 990 | 1155 | 1320 | µL |
**ICE COLD Fe-HBS **
- 3 rinses per coverslip
- 15 mL aliquots in the refrigerator
- need over 100 mL
HBS-paraformaldehyde 3.7%
- Need 60 mL
- Divided in 2 aliquots, one per fume hood
Ingredient | units | 10 | 15 | 20 | 25 | 40 | 50 | 60 |
---|---|---|---|---|---|---|---|---|
paraformaldehyde | g | 0.37 | .555 | 2 | 0.925 | 1.48 | 1.85 | 2.22 |
water | mL | 9 | 13.5 | 18 | 22.5 | 36 | 45 | 57.78 |
10X HBS | mL | 1 | 1.5 | 2 | 2.5 | 4 | 5 | 6 |
heat paraformaldehyde in water, then add 10X HBS
Add a drop of 10N NaOH
Heat and stir in the fume hood
6.2: Bulk endocytosis
6.2: Bulk endocytosis kdorfman Mon, 10/17/2011 - 17:05Bulk-phase Endocytosis
Cells for 6.2 2012
Cells for 6.2 2012 kdorfman Fri, 10/19/2012 - 12:323t3
2 coverslips per group
2 small diameter MatTeks per group
Cells for 6.2
Cells for 6.2 kdorfman Mon, 10/17/2011 - 17:09Lab is Monday 10/24/11
cells on coverslips: Students should use the same cell type as for Lab 6.1
- 3t3 8 (2 per group for 4 groups)
- LLCPk "
cells on MatTek dishes
(small diameter, so 100 µL will be enough to treat the cells):
- 3t3 8 (2 per group for 4 groups)
- LLCPk "
Reagents for 6.2
Reagents for 6.2 kdorfman Mon, 10/17/2011 - 20:29Monday 10/24
Students use the same cell type as for Lab 6.1
PBS for rinsing live cells WARM
PBS for rinsing fixed cells RT
Take from the supply of sterile aliquots
Growth media for incubation WARM
10 mL aliquots each
- F10-Hams for LLCPk cells
- DMEM for 3t3 cells
- non-CO2 medium for observing live cells (could use this for all cells for simplicity's sake)
TMR-dextran to stain endosomes in cells on coverslips
Invitrogen D3308, 10 mg
Make 10 mg/mL stock (add 1 mL to the bottle)
Make 8.8 µL aliquots in 2 mL tubes.
TMR-dextran Working concentration = 0.05 mg/mL in medium
2 coverslips at 50 µL/coverslip (=~110 µL/group)
Add 1751.2 µL medium to 8.8 µL 10mg/mL stock to make 0.05 mg/mL working solution.
Aliquot ~105 µL/group
#aliquots | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
---|---|---|---|---|---|---|---|---|---|
vi (10 mg/mL TMR-dex) | 1.1 | 2.2 | 3.3 | 4.4 | 5.5 | 6.6 | 7.7 | 8.8 | µL |
medium | 218.9 | 437.8 | 656.7 | 875.6 | 1094.5 | 1313.4 | 1532.3 | 1751.2 | µL |
vf | 220 | 440 | 660 | 880 | 1100 | 1320 | 1540 | 1760 | µL |
TMR-dextran/ FITC-dextran to stain endosomes in cells in MatTeks
tmr as above
FITC-dextran Working concentration = 0.5 mg/mL in medium
(1mg/mL might be better, but that uses up a whole bottle.)
2 dishes at 100 µL/dish (=~220 µL/group)
Add 1742.4 µL medium to an 8.8 µL aliquot of 10 mg/mL TMR-dex plus 8.8 µL 100 mg/mL FITC-Dex
#aliquots | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
---|---|---|---|---|---|---|---|---|---|
vi (10 mg/mL TMR-dex) | 1.1 | 2.2 | 3.3 | 4.4 | 5.5 | 6.6 | 7.7 | 8.8 | µL |
vi (100 mg/mL FITC-dex) | 1.1 | 2.2 | 3.3 | 4.4 | 5.5 | 6.6 | 7.7 | 8.8 | µL |
medium | 217.8 | 435.6 | 650.1 | 871.2 | 1089 | 1306.8 | 1524.6 | 1742.4 | µL |
vf | 220 | 440 | 660 | 880 | 1100 | 1320 | 1540 | 1760 | µL |
Methylamine solution (1M)
Sigma 426466-100mL 40% w/vol (=12.9M; MW=31) ~$25
(Working concentration ~20mM - ~20µL/mL. Students need ~100 µL)
Make 1 mL, aliquot ~120 µL
Solution | stock conc | final conc | 1 | 5 | 10 | 15 | 20 | mL |
---|---|---|---|---|---|---|---|---|
10X PBS | 10 | 1 | 0.1 | 0.5 | 1 | 1.5 | 2 | mL |
methylamine | 12.9 | 1 | 78 | 388 | 775 | 1163 | 1550 | µL |
plus water to final volume
Fixative
- PBS-paraformaldehyde 3.7%
- Need 70 mL
- Divided in 2 aliquots, one per fume hood
Ingredient | 10 | 15 | 20 | 25 | 40 | 50 | 60 | 70 | mL final |
---|---|---|---|---|---|---|---|---|---|
paraformaldehyde | 0.37 | 0.555 | 2 | 0.925 | 1.48 | 1.85 | 2.22 | 2.59 | g |
water | 9 | 13.5 | 18 | 22.5 | 36 | 45 | 57.78 | 63 | mL |
10X PBS | 1 | 1.5 | 2 | 2.5 | 4 | 5 | 6 | 7 | mL |
heat paraformaldehyde in water, then add 10X HBS
Add a drop of 10N NaOH
Heat and stir in the fume hood
OR
Ingredient | 10 | 15 | 20 | 25 | 40 | 50 | 60 | 70 | mL final |
---|---|---|---|---|---|---|---|---|---|
32% paraformaldehyde | 1.16 | 1.73 | 2.31 | 2.89 | 4.63 | 5.78 | 6.94 | 8.09 | mL |
10X PBS | 1 | 1.5 | 2 | 2.5 | 4 | 5 | 6 | 7 | mL |
water | 7.84 | 11.77 | 15.69 | 19.61 | 31.38 | 39.22 | 47.06 | 54.91 | mL |
6.3: Endosome txport
6.3: Endosome txport kdorfman Wed, 10/19/2011 - 18:44Transport of Endosomes along Microtubules
Wednesday 10/26/11
Cells for 6.3
Cells for 6.3 kdorfman Wed, 10/19/2011 - 18:47LLCPk-GFP-tub
3 small diameter MatTek dishes per group
Plate 24 dishes 2 days before lab
Reagents for 6.3
Reagents for 6.3 kdorfman Wed, 10/19/2011 - 18:48HBS-BSA 1mg/mL
to mix transferrin ( 3 mL) and nocodazole (15 mL) in
to rinse growth medium off before and after treatment. 6 mL per dish x 3 dishes per group x 7 groups = 126mL. Aliquot 20 mL.
make 200 mL
ingredient | 10 | 20 | 25 | 30 | 50 | 75 | 100 | 200 | 250 | mL |
---|---|---|---|---|---|---|---|---|---|---|
10 x HBS | 1 | 2 | 2.5 | 3 | 5 | 7.5 | 10 | 20 | 25 | mL |
BSA | 0.01 | 0.02 | 0.025 | 0.03 | 0.05 | 0.075 | 0.1 | 0.2 | 0.25 | g |
plus water to final volume
Fe-HBS-BSA
to mix transferrin in
Need 2.3 mL; make 3 mL
Solution | stock conc | final conc | 1 | 2 | 2.5 | 3 | 4 | mL |
---|---|---|---|---|---|---|---|---|
Fe-citrate (mM) | 89.4 | 0.1 | 1.118 | 2.237 | 2.796 | 3.35 | 4.47 | µL |
in Fe-HBS-BSA to final volume
Transferrin in Fe-HBS-BSA
Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD)
stock is 5 mg/ml (=62.5 µM).
Final concentration = 1 µM.
- Each group may do up to 3 100 µL treatments (= 3 x 7 x 105 µL = 2.205 mL)
- Make 105 µL aliquots (21)
- Make 2.3 mL: 36.8 µL AF-488-Tf + 2.263 mL Fe-HBS-BSA
if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.
# aliquots | 1 | 2 | 3 | 6 | 9 | 12 | 15 | 18 | 21 | |
---|---|---|---|---|---|---|---|---|---|---|
vi 62.5µM tf | 1.68 | 3.36 | 5.04 | 10.1 | 15.1 | 20.2 | 25.2 | 30.2 | 35.3 | µL |
HBS-Fe-BSA | 103.3 | 206.6 | 310 | 620 | 930 | 1240 | 1550 | 1860 | 2170 | µL |
Vf 1 µM tf | 105 | 210 | 315 | 630 | 945 | 1260 | 1575 | 1890 | 2205 | µL |
Nocodazole
Acros 358240100, 10 mg
1µM in HBS-BSA
(Working concentration range is usually 100nM - 30 µM.)
Stock is 10 mg/mL (=33mM)
(Add 1mL DMSO to 10 mg in bottle = 33mM. Aliquot 3.03 µL. To make 1mM, add 97 µL.)
Make enough for each group to do two 1 mL treatments.
Aliquot 1.01 mL per tube, 15 tubes = 15 mL. Make 16 mL
- Add 97 µL HBS-BSA to 3 µL 33mM in tube to make 1 mM
- Mix 1 part 1mM with 999 parts HBS-BSA to make 1µM: 16 mL + 1.6 µL
# aliquots | 1 | 2 | 3 | 4 | 5 | 10 | 15 | |
---|---|---|---|---|---|---|---|---|
vi 1 mM noc | 1.01 | 2.02 | 3.03 | 4.04 | 5.05 | 10.1 | 15.15 | µL |
HBS-BSA | 1.009 | 2.018 | 3.027 | 4.036 | 5.045 | 10.09 | 15.135 | mL |
vf 1 µM noc | 1.01 | 2.02 | 3.03 | 4.04 | 5.05 | 10.1 | 15.15 | mL |
non-CO2 medium
to mix TMR-dextran in
to incubate cells in
TMR-dextran
in non-CO2 medium
Each group may do up to 3 treatments = 3 x 7 x 100 µL = 2.205 mL.
Invitrogen D3308, 10 mg
Make 100 mg/mL stock (add 100µL to the bottle)
1 µL aliquots
TMR-dextran Working concentration = 0.05 mg/mL in medium
100 µL per treatment
Aliquot 105 µL, make enough for each group to do 3
Make 10 mg/mL dilution to make student aliquots
1 µL 100 mg/mL stock + 9µL medium
no. aliquots | 1 | 2 | 3 | 6 | 9 | 12 | 15 | 18 | 21 | |
---|---|---|---|---|---|---|---|---|---|---|
vi (10 mg/mL TMR-dex) | 0.525 | 1.05 | 1.575 | 2.2 | 3.15 | 4.2 | 6.3 | 7.875 | 11.025 | µL |
medium | 104.5 | 128.9 | 656.7 | 875.6 | 1094.5 | 1313.4 | 1532.3 | 1751.2 | µL | |
vf (0.05 mg/mL TMR-dex) | 105 | 210 | 315 | 630 | 945 | 1260 | 1575 | 1890 | 2205 | µL |
7.2: Calcium
7.2: Calcium kdorfman Wed, 10/26/2011 - 19:05Release of Internal Calcium Stores
Wednesday, 11/2/11
Rescheduled for Monday, 11/7/11, due to snowday 10/31/11
7.1: External stimuli
7.1: External stimuli kdorfman Wed, 10/26/2011 - 16:08Cellular Responses to External Stimuli
Monday, 10/31/11
Rescheduled for 11/2/11 because of storm on 10/31/11
Cells for 7.1
Cells for 7.1 kdorfman Wed, 10/26/2011 - 16:123t3 on large diameter MatTek dishes
4 dishes per group plus extra = 32
Plate 2 days before class.
Reagents for 7.1
Reagents for 7.1 kdorfman Wed, 10/26/2011 - 16:13HBS
HBS kdorfman Wed, 10/26/2011 - 16:15HBS for washes and making solutions
25 mL per group
(total = 175 mL)
Make from 10X
Make 350 mL
Pluronic F-127
Pluronic F-127 kdorfman Wed, 10/26/2011 - 16:26Stock
comes as dry granules - store on shelf
Also comes as solution Fisher 50-310-493
PROMOCELL GMBH PLURONICF-127 20%IN DMSO 1ML $44
Make 0.5 mL with 0.1g (= 200 mg/mL =20% w/v) in DMSO
10 µL aliquots - each makes 10 mL 0.02%
Store in freezer.
ATP
ATP kdorfman Wed, 10/26/2011 - 17:1310 mM ATP stock (from Maniotis)
60 mg ATP in 8 mL H2O
pH to 7.0 with 0.1 M NaOH
Bring volume to 10 mL with H2O
Aliquot and freeze.
10 µM solution for use (=5.731 mg/L) in HBS
Make enough for Labs 7.1 and 7.2:
Lab 7.1: 0.750 mL per coverslip x 4 coverslips x 7 groups = 21 mL
Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL
Make 40 mL
40 µL 10 mM ATP stock in 40 mL HBS
(1 µL 10 mM ATP stock per 1 mL HBS.)
Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2
Bradykinin
Bradykinin kdorfman Wed, 10/26/2011 - 17:221 mg/mL stock
(= 1.0602 mM)
Sigma B3259 - 1 mg septum bottle
MW = 1060.2
Add 1 mL H2O to the 1 mg in the bottle (NO CALCIUM! - some will be used for the calcium experiments in Lab 7.2)
Aliquots are 21.2 µL.
2 µM solution for use
(=2.12 µg/mL)
NOTE: Rather weak response in 2011. Try doubling the concentration?
Make enough for Labs 7.1 and 7.2:
- Lab 7.1: 0.750 mL per coverslip x 4 coverslips x 7 groups = 21 mL
- Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL
Make 40 mL
~2 µL 1 mg/mL stock per 1 mL working solution
Each 21.2 µL aliquot of 1 mg/mL makes 10 mL 2µM working solution
84.8 µL 1 mg/mL stock in 40 mL HBS
Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2)
4 µM solution for use
(=4.24 µg/mL)
NOTE: Double the 2011 concentration
Make enough for Labs 7.1 and 7.2:
- Lab 7.1: 0.750 mL per coverslip x 4 coverslips x 7 groups = 21 mL
- Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL
Make 40 mL
~4 µL 1 mg/mL stock per 1 mL working solution
Each 21.2 µL aliquot of 1 mg/mL makes 5 mL 2µM working solution
169.6 µL 1 mg/mL stock in 40 mL HBS (8 aliquots)
Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2)
Fluo-4
Fluo-4 kdorfman Wed, 10/26/2011 - 16:22Fluo-4 stock
Invitrogen F14217 500 µL
1mM in DMSO
Protect from light
Store in dissicator.
20 µL aliquots. Each makes 10 mL of 2 µM solution
Fluo-4 staining solution
2 µM Fluo-4 + 0.02% pluronic in HBS
Make enough for 7.1 and 7.2: 250 µL x 4 expts x 8 grps x 2 days = 16 mL.
Make 20 mL
Need 16 aliquots, each ~1.05 mL
ingredient | 5 | 10 | 15 | 20 | mL |
---|---|---|---|---|---|
1 mM Fluo-4 stock | 10 | 20 | 30 | 40 | µL |
20 % w/v Pluoronic | 5 | 10 | 15 | 20 | µL |
plus HBS to final volume
Vasopressin
Vasopressin kdorfman Wed, 10/26/2011 - 18:291 mM stock in water
(NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)
1 mg in bottle, MW = 1084
Add 0.92 mL to make 1mM stock
1 µL aliquots in freezer
1 µM dilution to make student solutions
1 µL 1 mM in 999 µL water
(NO CALCIUM, so stock can be used for Ca-free solutions in Lab 7.2)
10 nM (= 1.084 µg/mL) for students
NOTE: Rather weak response in 2011. Try doubling the concentration?
1 part 1µM dilution to 99 parts HBS
Make enough for Labs 7.1 and 7.2: Lab 7.1: 0.750 mL per coverslip x 4 coverslips x 7 groups = 21 mL Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL
Make 40 mL: 400 µL 1µM Vasopressin + 39.6 mL HBS
Make 24 aliquots of 1.6 mL (16 for Lab 7.1, 8 for Lab 7.2
test drop downs for 7.1 reagents
test drop downs for 7.1 reagents kdorfman Thu, 10/27/2011 - 16:55This page tests the use of collapsible sections for recipe pages
Cells for 7.2
Cells for 7.2 kdorfman Wed, 10/26/2011 - 19:073t3 on large-diameter MatTek dishes
4 dishes per group, plus extra = 32
Plate on Monday before Wednesday lab, or Saturday if it's on Monday
Reagents for 7.2
Reagents for 7.2 kdorfman Wed, 10/26/2011 - 19:08Use left-over aliquots of stimulants from 7.1 with calcium, make calcium-free reagents for 7.2
ATP
ATP kdorfman Wed, 10/26/2011 - 19:2310 mM ATP Stock
(from Maniotis)
60 mg ATP in 8 mL H2O
pH to 7.0 with 0.1 M NaOH
Bring volume to 10 mL with H2O
Aliquot and freeze.
10 µM (=5.731 mg/L) ATP in HBS
Use ATP aliquots from Lab 7.1
10 µM ATP, Calcium-free
Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL
Make 13 mL
13 µL 10 mM ATP stock in 13 mL ca- HBS (1 µL 10 mM ATP stock per 1 mL HBS.)
8 1.6 mL aliquots for Lab 7.2
Bradykinin
Bradykinin kdorfman Wed, 10/26/2011 - 19:251mg/mL Bradykinin stock
(= 1.0602 mM) Sigma B3259 - 1 mg septum bottle
MW = 106.02
Add 1 mL H2O to the 1 mg in the bottle (NO CALCIUM! - some will be used for the calcium experiments in Lab 7.2)
2 µM Bradykinin in HBS
Use bradykinin aliquots from Lab 7.1
2 µM Bradykinin, Calcium-free
(=2.12 µg/mL)
Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL
Frozen aliquots are 21.2 µL 1 mg/mL. Makes 10 mL
~2 µL 1 mg/mL stock per 1 mL working solution
21.2 µL 1 mg/mL stock in 10 mL Ca-free HBS
Make 6 aliquots of 1.6 mL
Make more if necessary. (Aliquots should have been enough for 15 mL?)
Fluo-4
Fluo-4 kdorfman Wed, 10/26/2011 - 19:17Use Fluo-4 aliquots from Lab 7.1
HBS
HBS kdorfman Wed, 10/26/2011 - 19:14HBS needed for rinsing and to make other solutions
(3 rinses + 1 sham dilution) x 4 expts/grp x 1 mL = 16 mL
Aliquot 25 mL per group
300 mL HBS
300 mL Ca-free HBS
Ionomycin
Ionomycin kdorfman Wed, 10/26/2011 - 19:41Ionomycin stock
1 mM in DMSO or EtOH. 1 mg makes 1.34 mL stock.
3 µM Ionomycin in HBS
Dilute stock 1:333 in HBS: 3 µL/1 mL
21 µL in 7 mL, aliquot 800 µL
They only need 750µL, because they do one experiment in HBS, then two in Ca-free HBS.
3 µM Ionomycin in Ca-free HBS
Dilute stock 1:333 in Ca-free HBS: 3 µL/1 mL
39 µL in 13 mL
Aliquot 1.55 mL so they can do two 750 µL experiments
Vasopressin
Vasopressin kdorfman Wed, 10/26/2011 - 19:341 mg/mL (= 0.9225 mM) Vasopressin stock in water
1 µM dilution to make student solutions
1 µL 1 mg/mL in 922.5 µL water (NO CALCIUM, so stock can be used for Ca-free solutions )
10 nM (= 1.084 µg/mL) Vasopressin in HBS for students
Use Vasopressin aliquots from Lab 7.1
Calcium Free Vasopressin 10 nM
10 nM (= 1.084 µg/mL) for students
1 part 1µM dilution to 99 parts Ca-free HBS
Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 7 groups = 10.5 mL
Make 13 mL: 130 µL + 13 mL Ca-free HBS
Make 8 aliquots of 1.6 mL
8: Projects
8: Projects kdorfman Tue, 11/22/2011 - 14:20Projects
Have students make a class poster for the bulletin board in the hall.
Chi-Square calculator
Chi-Square calculator kdorfman Fri, 12/21/2012 - 19:11ER-tracker
ER-tracker kdorfman Tue, 11/22/2011 - 14:22ER-Tracker from Invitrogen
E34250 ER-Tracker™ Red (BODIPY® TR glibenclamide) for live-cell imaging 100 μg
1mM Stock
Add 110 µL DMSO to the 100 µg in vial.
Aliquot 1 µL
Freeze
1 µM working concentration
- add 0.999 mL HBSS/Ca/Mg
Protocol
rinse with warm HBSS
treat with 1 mL warm 1 µM ER-tracker
incubate 15-30 min at 37C
To view stained cells
rinse with medium
view with fluorescence microscope
To fix cells
4% formaldehyde, for 2 min, at 37C
rinse 2x in buffer - NO TRITON X-100
HBSS/Ca/Mg
HBSS/Ca/Mg kdorfman Wed, 11/23/2011 - 20:08Hank's Balanced Salt Solution
To make 1 Liter from dry ingredients
Components | MW | (mg/L) | mM |
---|---|---|---|
Calcium Chloride (CaCl2) (dihyd.) | 147 | 185 | 1.26 |
Magnesium Chloride (MgCl2-6H2O) | 203 | 100 | 0.493 |
Magnesium Sulfate (MgSO4-7H2O) | 246 | 100 | 0.407 |
Potassium Chloride (KCl) | 75 | 400 | 5.33 |
Potassium Phosphate monobasic (KH2PO4) | 136 | 60 | 0.441 |
Sodium Bicarbonate (NaHCO3) | 84 | 350 | 4.17 |
Sodium Chloride (NaCl) | 58 | 8000 | 137.93 |
Sodium Phosphate dibasic (Na2HPO4-6H20) | 268 | 90.6 | 0.338 |
D-Glucose (Dextrose) | 180 | 1000 | 5.56 |
Filter sterilize
To make from stock solutions:
Components | Ci (M) | cf (mM) | 1000 | 500 | 250 | mL |
---|---|---|---|---|---|---|
CaCl2 | 0.5 | 1.26 | 2.52 | 1.26 | 0.63 | mL |
MgCl2 | 1 | 0.493 | 0.493 | 0.247 | 0.108 | mL |
MgSO4 | 1 | 0.407 | 0.407 | 0.204 | 0.102 | mL |
KCl | 1 | 5.33 | 5.33 | 2.67 | 0.131 | mL |
KH2PO4 | 1 | 0.441 | 0.441 | 0.22 | 0.11 | mL |
NaHCO3 | 0.5 | 4.17 | 8.34 | 4.17 | 0.209 | mL |
NaCl | 5 | 137.93 | 27.59 | 13.79 | 6.9 | mL |
Na2HPO4 | 1 | 0.338 | 0.338 | 0.169 | 0.085 | mL |
Glucose | - | - | 1 | 0.5 | 0.25 | g |
Filter sterilize
Latrunculin B
Latrunculin B kdorfman Wed, 11/30/2011 - 18:21Alexis Biochemicals 350-036-C100 Catalog # t110
Inhibits actin polymerization and disrupts microfilament organization, as well as microfilament-mediated processes
Reported to be 10 to 100-fold more potent than the cytochalasins. May act more slowly, though. Lots of variation between cell lines.
Inactivated by FBS. (Rinse medium off before treatment.)
MW = 395.5
Soluble in DMSO and ethanol.
Store in freezer.
Active concentration range: 90 nm ( =~0.1µM) to 2.5 µM
(2.5 µM = 25 x 100 nm)
Stock is 1mm
µL 1mm stock | µL serum-free buffer or medium | µM final concentration |
---|---|---|
1 * | 99 | 10 |
1 | 399 | 2.5 |
1 | 999 | 1 |
*Use this to make further dilutions
Projects 2012
Projects 2012 kdorfman Wed, 11/07/2012 - 20:29For Wed, 11/14/12
Names | cell type | # dish/coverslip | reagents |
---|---|---|---|
Jonathan & Laura | LL parentals | 5 slips | fixative, mounting medium |
Mike & Theresa | 3t3 | 4 dishes | transferrin, Fe-HBS, HBS |
Mike & Mike | LL alpha-GFP, myo-RFP | 4 dishes | blebbistatin, non-CO2 medium |
Cassie & Marc | LL parentals | 4 dishes | nuc-blue, HBS, Fluo-4, non-CO2 |
Dave & Tyler | LL alpha-GFP | 2 dishes, 2 slips | Fix, mitotracker, Rhodamine123, non-CO2, PBS |
Mike & Katherine | LL parentals | 3 dishes | ceramide, non-CO2, PBS |
Ray & Poya | LL alpha-GFP | 2 dishes | non- CO2 |
Plating:
- LL parental: 5 coverslips, 7 dishes
- LL alphas: 4 dishes, 2 coverslips
- LL alpha, myo: 4 dishes
- 3t3: 4 dishes
Blebbistatin
Blebbistatin kdorfman Wed, 11/21/2012 - 19:51(-)-Blebbistatin purchased from Sigma B05650-1mg
Info from Cayman:
A stock solution may be made by dissolving the (±)-blebbistatin in an organic solvent purged with an inert gas. (±)-Blebbistatin is soluble in organic solvents such as DMSO and dimethyl formamide (DMF). The solubility of (±)-blebbistatin in these solvents is approximately 10 mg/mL.
(±)-Blebbistatin is sparingly soluble in aqueous buffers. For maximum solubility in aqueous buffers, (±)-blebbistatin should first be dissolved in DMF and then diluted with the aqueous buffer of choice. (±)-Blebbistatin has a solubility of approximately 0.5 mg/ml in a 1:1 solution of DMF:PBS (pH 7.2) using this method. We do not recommend storing the aqueous solution for more than one day.
(±)-Blebbistatin is a selective cell-permeable inhibitor of non-muscle myosin II ATPases.1,2 It rapidly and reversibly inhibits Mg-ATPase activity and in vitro motility of non-muscle myosin IIA and IIB for several species (IC50 = 0.5-5.0 μM), while poorly inhibiting smooth muscle myosin (IC50 = 80 μM).3 Through these effects, blebbistatin blocks apoptosis-related bleb formation, directed cell migration and cytokinesis in vertebrate cells. Blebbistatin is inactivated by UV light,4 which may be particularly important in fluorescent cell imaging applications.
Note green fluorescent crystals seen in a 75µM solution.
Stock was 1 mg/mL in DMSO
44 µL 1mg/mL blebbistatin in DMSO in 1 mL non-CO2 medium
REDO
1 mg in 34 µL DMSO = ~100mM
MW = 292
Use at ~150 µM
Cells Monday 11/26
Cells Monday 11/26 kdorfman Mon, 11/19/2012 - 19:20Names | cell type | dishes | coverslips | reagents |
---|---|---|---|---|
Jonathan & Laura | LL parentals | 5 | (Sunday) | |
Mike & Theresa | 3t3 | 6 | ||
Mike & Mike | LL gamma tub | 4 | ||
Cassie & Marc | LL parentals | 4 | fluo4-AM | |
Dave & Tyler | LL alpha-GFP | 2 | 2 | |
Mike & Katherine | LL parentals | 2 | ||
LL alpha | 2 | |||
Ray & Poya | LL alpha-GFP | 8 | ciliobrevin (HPI-4) (fix) BI2536 |
Plating:
- LL parental:
- 5 coverslips,
- 6 dishes
- LL gamma: 4 dishes
- LL alpha:
- 2 dishes
- 10 coverslips
- LL alpha, myo:
- 3t3: 6 dishes
Cells Wed 11/14
Cells Wed 11/14 kdorfman Mon, 11/19/2012 - 19:41For Wed, 11/14/12
Names | cell type | # dish/coverslip | reagents |
---|---|---|---|
Jonathan & Laura | LL parentals | 5 slips | fixative, mounting medium |
Mike & Theresa | 3t3 | 4 dishes | transferrin, Fe-HBS, HBS |
Mike & Mike | LL alpha-GFP, myo-RFP | 4 dishes | blebbistatin, non-CO2 medium |
Cassie & Marc | LL parentals | 4 dishes | nuc-blue, HBS, Fluo-4, non-CO2 |
Dave & Tyler | LL alpha-GFP | 2 dishes, 2 slips | Fix, mitotracker, Rhodamine123, non-CO2, PBS |
Mike & Katherine | LL parentals | 3 dishes | ceramide, non-CO2, PBS |
Ray & Poya | LL alpha-GFP | 2 dishes | non- CO2 |
Plating:
- LL parental: 5 coverslips, 7 dishes
- LL alphas: 4 dishes, 2 coverslips
- LL alpha, myo: 4 dishes
- 3t3: 4 dishes
Cells Wed 11/28
Cells Wed 11/28 kdorfman Mon, 11/26/2012 - 19:48Names | cell type | dishes | coverslips | reagents |
---|---|---|---|---|
Jonathan & Laura | 0 | 0 | 0 | |
Mike & Theresa | 3t3 | 3 normal and 6 light (for weekend) | ||
Mike & Mike | LL gamma tub | 4 | Blebbistatin | |
Cassie & Marc | LL parentals | 2 | (fluo4-AM) | |
Dave & Tyler | LL alpha-GFP | 2 | 2 | |
Mike & Katherine | - | 0 | 0 | |
Ray & Poya | 0 | 0 | 0 |
HPI-4 (ciliobrevin)
HPI-4 (ciliobrevin) kdorfman Tue, 11/20/2012 - 15:51Sigma H4541-5MG
MW 358.18
solubility 20 mg/mL
Make stock in DMSO
100mM = 5 mg/140 µL
http://www.ncbi.nlm.nih.gov/pubmed/22425997#
Use at 100uM. 1 µL/1 mL
light sensitive
fix in para glut, to preserve the GFP micro tubules.
Para formaldehyde may also work
cells Mon 12/3
cells Mon 12/3 kdorfman Wed, 11/28/2012 - 20:05Names | cell type | dishes | coverslips | reagents |
---|---|---|---|---|
Jonathan & Laura | B16 | - | 5 | formaldehyde fix |
Mike & Theresa | 3t3 | 6 | 0 | |
Mike & Mike | LL gamma tub | 4 | - | (blebbistatin) |
Cassie & Marc | LL parentals | 4 | 0 | |
Dave & Tyler | LL actin-GFP | 2 | - | |
Mike & Katherine | LL alpha tub | 0 | 3 | formaldehyde fix |
Ray & Poya | - | - | - |
Thaw B16 and actins on Thursday 11/29
Plate all types lightly on Friday 11/30 for Monday
cells Sat 12/1
cells Sat 12/1 kdorfman Wed, 11/28/2012 - 20:16Names | cell type | dishes | coverslips | reagents |
---|---|---|---|---|
Mike & Theresa | 3t3 | 3 | - | |
Cassie & Marc | LL parentals | 6 | - | (fluo4-AM) |
Plate on Thursday
Also make heavy flasks for Plating on Friday
cells Wed 12/5
cells Wed 12/5 kdorfman Mon, 12/03/2012 - 16:36Names | cell type | dishes | coverslips | reagents |
---|---|---|---|---|
Jonathan & Laura | ||||
Mike & Theresa | 3t3 (light) | 4 | ||
Mike & Mike | gamma tubulin | 4 | 0 | |
Cassie & Marc | Parental LLCPK1 (heavy) | 6 | 0 | HBS! |
Dave & Tyler | ||||
Mike & Katherine | ||||
Ray & Poya |
projects 2011
projects 2011 kdorfman Wed, 11/07/2012 - 20:15Cells for 11/28/11
Cells for 11/28/11 kdorfman Wed, 11/23/2011 - 18:06Group | Cells | MatTek | Coverslip | Reagents |
---|---|---|---|---|
Kevin & Chris | LLCPk | 0 | 6 | ER tracker, anti golgi |
Megan & Olivia | actin | 3 (20 mm) | 0 | cyto D |
Andrew & Amelia | actin | 4 (20 mm) | 0 | Cyto D |
Alex & Quentin | tubulin | 4 (14mm) | 0 | Noc & taxol |
Luke & Don | tubulin | 4 (20 mm) | 0 | ER tracker |
Jon & Silas | 3t3 | 2 (20 mm) | 4 | Cyto D |
Molly & Jason | s2 | 0 | 0 | Con A, colcemid or colchicine |
Cells for 11/30/11
Cells for 11/30/11 kdorfman Mon, 11/28/2011 - 15:13Group | Cells | MatTek | Coverslip | Reagents |
---|---|---|---|---|
Kevin & Chris | LLCPk | 4 | ER tracker, anti golgi | |
Meghan & Olivia | actin | 7 (20 mm) | cyto D, latrunculin | |
Andrew & Amelia | actin | 5 (20 mm) | Cyto D | |
Alex & Quentin | tubulin | 4 (14mm) | Noc & taxol | |
Luke & Don | tubulin | 4 (20mm) | ER tracker, noc | |
Jon & Silas | 3t3 | 5 (20mm) | Cyto D, ATP | |
Molly & Jason | s2 | Con A, colcemid or colchicine |
Cells for 12/5/11
Cells for 12/5/11 kdorfman Wed, 11/30/2011 - 21:11Group | Cells | MatTek | Coverslip | Reagents |
---|---|---|---|---|
Kevin & Chris | LLCPk | 4 | ER tracker, anti golgi | |
Meghan & Olivia | actin | 5 (20 mm) | cyto D, latrunculin | |
Andrew & Amelia | actin | 2 (20 mm) | 6 | Cyto D |
Alex & Quentin | tubulin | 4 (14mm) | Noc & taxol | |
Luke & Don | tubulin | 4 (20mm) | ER tracker, noc | |
Jon & Silas | 3t3 | (20mm) | 5 | Cyto D, ATP |
Molly & Jason | s2 | Con A, colcemid or colchicine |
Cells for 12/7
Cells for 12/7 kdorfman Mon, 12/05/2011 - 17:26Group | Cells | MatTek | Coverslip | Reagents |
---|---|---|---|---|
Kevin & Chris | LLCPk | ER tracker, anti golgi | ||
Meghan & Olivia | actin | (20 mm) | cyto D, latrunculin | |
Andrew & Amelia | actin | (20 mm) | Cyto D | |
Alex & Quentin | tubulin | (14mm) | Noc & taxol | |
Luke & Don | tubulin | (20mm) | ER tracker, noc | |
Jon & Silas | 3t3 | (20mm) | Cyto D, ATP | |
Molly & Jason | s2 | Con A, colcemid or colchicine |
Labs 2013
Labs 2013 kdorfman Thu, 09/26/2013 - 14:271.1 2013
1.1 2013 kdorfman Thu, 09/26/2013 - 14:28W 9/04
Intro to Equipment
1.2 2013
1.2 2013 kdorfman Thu, 09/26/2013 - 14:28W 9/09
Imaging mitosis with fixed cells. Image Spatial Quantification
1.3 2013
1.3 2013 kdorfman Thu, 09/26/2013 - 14:32W 9/11
Image Formation in the Microscope
2.1 2013
2.1 2013 kdorfman Thu, 09/26/2013 - 14:33M 9/16
Numerical Aperture
2.2 2013
2.2 2013 kdorfman Thu, 09/26/2013 - 14:37W 9/18
Resolution
3.1 2013
3.1 2013 kdorfman Thu, 09/26/2013 - 14:38M 9/23
Fluorescence Microscopy
3.2 2013
3.2 2013 kdorfman Thu, 09/26/2013 - 14:39W 9/25
Identification of Cellular Organelles
3.3 2013
3.3 2013 kdorfman Thu, 09/26/2013 - 14:40M 9/30
Labeling Cellular Structures
Cells
paraglut fixed LL's for students to stain for tubulin (make extra!)
paraglut fixed LL's for students to stain for phalloidin
paraglut fixed 3t3's for students to stain for phalloidin
live LL's to fix in paraglut (2014: treat first with mitotracker, then fix in formaldehyde no detergent)
Solutions
PBS to rinse coverslips
2% PBS-BSA to make up antibodies
Materials
forceps
50 mL beakers (switch to holders and 100 mL beakers?)
petri dishes for humid chambers
filter paper for humid chambers
mounting medium
slides
nail polish
kimwipes
parafilm
3.4 2013
3.4 2013 kdorfman Thu, 09/26/2013 - 14:41W 10/2
Imaging Living Cells Expressing GFP-Tagged Proteins
LL-GFP-tubulin
LL-GFP-actin
LL nucleofected with plasmids
- H2B
2014
LL-EB1-GFP stable line
LL-alpha tubulin=GFP stable line
** transfected**
H2B m cherry
alpha actininin (GFP?)
4.1 2013
4.1 2013 kdorfman Thu, 09/26/2013 - 14:42W 10/9
Motility in Control Cells
On MatTek dishes:
3t3
B16
LL-GFP actin
Non-CO2 medium
4.2 2013
4.2 2013 kdorfman Thu, 09/26/2013 - 14:43Tu 10/15 & W 10/16
Mechanism of Motility
Students check for effect of dose and time on actin filaments. Treat LL parentals with cytochalasin D, then stain actin with phalloidin.
LL on coverslips
non-CO2 medium
5.1 2013
5.1 2013 kdorfman Thu, 09/26/2013 - 14:45W 10/23
Receptor-mediated endocytosis
NO LL's
NO high iron
3 coverslips of 3t3 per group
Try Lysotracker (Invitrogen L-7528)
50 - 75 nM
stock = 1 mM in DMSO
30 min - 2 hours incubation warm.
Add lysotracker to cells at 1 pm.
- Make 100 µM stock
- 1 µL of 100 µM stock to each dish. Assume ~1.5 mL per dish
Omit the high-iron treatment
For convenience, all staining, incubation, etc. will be done in HBS.
Each group does 3 coverslips of 3t3.
HBS to mix everything else in. There is a bottle of sterile 10X on the prep room bench.
- 6 mL aliquots COLD (final rinse before fix coverslips 1-3)
- 15 mL aliquots RT in hood (to rinse off fixative)
- Need ~300 mL total
Fe-HBS
- 20 mL aliquots COLD to rinse off growth medium, then tf
- 5 mL aliquots WARM (incubation after tf treatment)
- need some to make Fe-HBS-BSA for transferrin
- make 200 mL
Solution | stock conc | final conc | 25 | 50 | 75 | 100 | 200 | mL |
---|---|---|---|---|---|---|---|---|
10X HBS | 10 | 1 | 2.5 | 5 | 7.5 | 10 | 20 | mL |
Fe-citrate (mM) | 89.4 | 0.1 | 27.96 | 55.92 | 84 | 112 | 224 | µL |
plus water to final volume
Fe-HBS-BSA 1 mg/mL to make transferrin solution
- 5 mg BSA in 5 mL Fe-HBS
Transferrin in Fe-HBS-BSA
- Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM). Final concentration = 1 µL.
- Each group does 3 50-µL treatments (need extra for pipetting error)
- Make 1.2 mL: 19.2 µL AF-488-Tf + 1.1808 mL Fe-HBS-BSA
- Aliquot 165 µL
if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.
# aliquots | 3 | 6 | 9 | 12 | 15 | 18 | 21 | 24 | |
---|---|---|---|---|---|---|---|---|---|
vi (Tf) | 2.64 | 5.28 | 7.92 | 10.56 | 13.2 | 15.84 | 18.48 | 21.12 | µL |
HBS | 162.4 | 324.7 | 487.1 | 649.4 | 811.8 | 974.2 | 1136.5 | 1298.9 | µL |
vf | 165 | 330 | 495 | 660 | 825 | 990 | 1155 | 1320 | µL |
-
- Need 60 mL
- Divided in 2 aliquots, one per fume hood
Old fashioned way:
Ingredient | units | 10 | 15 | 20 | 25 | 40 | 50 | 60 |
---|---|---|---|---|---|---|---|---|
paraformaldehyde | g | 0.37 | .555 | 0.74 | 0.925 | 1.48 | 1.85 | 2.22 |
water | mL | 9 | 13.5 | 18 | 22.5 | 36 | 45 | 57.78 |
10X HBS | mL | 1 | 1.5 | 2 | 2.5 | 4 | 5 | 6 |
heat paraformaldehyde in water, then add 10X HBS
Add a drop of 10N NaOH
Heat and stir in the fume hood
OMIT HIGH IRON
Solution | stock conc | final conc | 10 | 15 | 20 | 25 | 30 | 50 | mL |
---|---|---|---|---|---|---|---|---|---|
10X HBS | 10 | 1 | 1 | 1.5 | 2 | 2.5 | 3 | 5 | mL |
Fe-citrate (mM) | 89.4 | 2 | 223.7 | 335.6 | 448 | 559.2 | 671 | 1118.4 | µL |
plus water to final volume
5.2 2013
5.2 2013 kdorfman Thu, 09/26/2013 - 14:46M 10/28
Bulk-phase endocytosis
** 2 coverslips, 2 small diameter MatTek dishes 3t3
PBS for rinsing live cells WARM
PBS for rinsing fixed cells RT
Take from the supply of sterile aliquots
Growth media for incubation WARM
10 mL aliquots each
- DMEM for 3t3 cells
- non-CO2 medium for observing live cells (could use this for all cells for simplicity's sake)
TMR-dextran to stain endosomes in cells on coverslips
Invitrogen D3308, 10 mg
Make 10 mg/mL stock (add 1 mL to the bottle)
Make 8.8 µL aliquots in 2 mL tubes.
TMR-dextran Working concentration = 0.05 mg/mL in medium
2 coverslips at 50 µL/coverslip (=~110 µL/group)
Add 1751.2 µL medium to 8.8 µL 10mg/mL stock to make 0.05 mg/mL working solution.
Aliquot ~105 µL/group
#aliquots | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
---|---|---|---|---|---|---|---|---|---|
vi (10 mg/mL TMR-dex) | 1.1 | 2.2 | 3.3 | 4.4 | 5.5 | 6.6 | 7.7 | 8.8 | µL |
medium | 218.9 | 437.8 | 656.7 | 875.6 | 1094.5 | 1313.4 | 1532.3 | 1751.2 | µL |
vf | 220 | 440 | 660 | 880 | 1100 | 1320 | 1540 | 1760 | µL |
TMR-dextran/ FITC-dextran to stain endosomes in cells in MatTeks
tmr as above
FITC-dextran Working concentration = 0.5 mg/mL in medium
(1mg/mL might be better, but that uses up a whole bottle.)
2 dishes at 100 µL/dish (=~220 µL/group)
Add 1742.4 µL medium to an 8.8 µL aliquot of 10 mg/mL TMR-dex plus 8.8 µL 100 mg/mL FITC-Dex
#aliquots | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
---|---|---|---|---|---|---|---|---|---|
vi (10 mg/mL TMR-dex) | 1.1 | 2.2 | 3.3 | 4.4 | 5.5 | 6.6 | 7.7 | 8.8 | µL |
vi (100 mg/mL FITC-dex) | 1.1 | 2.2 | 3.3 | 4.4 | 5.5 | 6.6 | 7.7 | 8.8 | µL |
medium | 217.8 | 435.6 | 650.1 | 871.2 | 1089 | 1306.8 | 1524.6 | 1742.4 | µL |
vf | 220 | 440 | 660 | 880 | 1100 | 1320 | 1540 | 1760 | µL |
Methylamine solution (1M)
Sigma 426466-100mL 40% w/vol (=12.9M; MW=31) ~$25
(Working concentration ~20mM - ~20µL/mL. Students need ~100 µL)
Make 1 mL, aliquot ~120 µL
Solution | stock conc | final conc | 1 | 5 | 10 | 15 | 20 | mL |
---|---|---|---|---|---|---|---|---|
10X PBS | 10 | 1 | 0.1 | 0.5 | 1 | 1.5 | 2 | mL |
methylamine | 12.9 | 1 | 78 | 388 | 775 | 1163 | 1550 | µL |
plus water to final volume
Fixative
- PBS-paraformaldehyde 3.7%
- Need 70 mL
- Divided in 2 aliquots, one per fume hood
Ingredient | 10 | 15 | 20 | 25 | 40 | 50 | 60 | 70 | mL final |
---|---|---|---|---|---|---|---|---|---|
paraformaldehyde | 0.37 | 0.555 | 2 | 0.925 | 1.48 | 1.85 | 2.22 | 2.59 | g |
water | 9 | 13.5 | 18 | 22.5 | 36 | 45 | 57.78 | 63 | mL |
10X PBS | 1 | 1.5 | 2 | 2.5 | 4 | 5 | 6 | 7 | mL |
heat paraformaldehyde in water, then add 10X HBS
Add a drop of 10N NaOH
Heat and stir in the fume hood
OR http://wahoo.nsm.umass.edu/content/paraformaldehyde-fix
Ingredient | 10 | 15 | 20 | 25 | 40 | 50 | 60 | 70 | mL final |
---|---|---|---|---|---|---|---|---|---|
32% paraformaldehyde | 1.16 | 1.73 | 2.31 | 2.89 | 4.63 | 5.78 | 6.94 | 8.09 | mL |
10X PBS | 1 | 1.5 | 2 | 2.5 | 4 | 5 | 6 | 7 | mL |
water | 7.84 | 11.77 | 15.69 | 19.61 | 31.38 | 39.22 | 47.06 | 54.91 | mL |
5.2 2014
5.2 2014 kdorfman Fri, 10/24/2014 - 19:07Cells
- 3t3 on coverslips - 2 per group
- 3t3 on MatTek dishes (small diameter)
- LLCPk GFP-alpha tubulin on coverslips 1 per group
- LL parentals on coverslips 2 per group
Reagents
TMR-dextran to stain endosomes in cells on coverslips
Invitrogen D3308, 10 mg
Make 10 mg/mL stock (add 1 mL to the bottle)
TMR-dextran Working concentration = 0.05 mg/mL in medium
- 2 coverslips at 50 µL/coverslip (=~110 µL/group) + 2 live cells at 100 µL/mattek
- Aliquot 5.2 µL 10 mg/mL
- Label says to add 98.8 µL to make 0.5mg/mL working soloution
Lysotracker (Invitrogen L-7528 - 20 x 50 µL)
- 50 - 75 nM
- stock = 1 mM in DMSO
- aliquot 10 µL in 2 mL tubes, so they can make 2 mL medium
- 30 min - 2 hours incubation warm.
Transferrin in Fe-HBS-BSA
- Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD) stock is 5 mg/ml (=62.5 µM). Final concentration = 1 µL.
- Each group does 2 50-µL treatments (need extra for pipetting error)
- Make 1.2 mL: 19.2 µL AF-488-Tf + 1.1808 mL Fe-HBS-BSA
Aliquot 165 µL
if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.
5.2 2015
5.2 2015 kdorfman Wed, 11/04/2015 - 14:50Cells
- 2 coverslips 3t3 per pair for TMR-dextran
- 2 LL-GFP-tubulin in MatTek dishes per pair for lysotracker & endosome movement
- 3t3 in MatTek for live studies
Reagents
HBS-BSA (1 mg/mL). per pair ~7mL:
- 6 mL for 3 rinses live cells
- 1 mL for nocodazole
- 0.1 mL for Transferrin
Nocodazole 1 µM in HBS-BSA 1 mL per pair
- aliquots are 3 µL 3mM in DMSO
- add 97 µL to make 1mM stock
- dilute 1:1000 in HBS-BSA (10 µL in 10 mL)
Transferrin in Fe-HBS-BSA to label live cells 100 µL/pair
- Stock is 5 mg/ml (=62.5 µM)
- Working conc = 1 µM
- 0.016 µL Tf/µL solution
- 1 mL = 16 µL Tf + 984 µL Fe-HBS-BSA
Lysotracker in non-CO2 medium 1mL/pair
- stock is 1 mM
- working solution is 50 - 75 nM
- Dilute 7.5:100,000 = 0.75 µL/10 mL
TMR-dextran (Invitrogen D3308, 10 mg) in non-CO2 medium
- 100 µL to stain in dish
- 100 µL to stain 2 coverslips
- 10 mg/mL stock, in 8.8 ML aliquots
- 0.05 mg/mL working concentration
- aliquot ~210 µL per pair
CO2 medium (to rinse and return to incubator)
Non-CO2 medium (for imaging)
PBS for rinsing
3.7% paraformaldehyde in PBS for fixation: 3 mL/pair
5.3 2013
5.3 2013 kdorfman Thu, 09/26/2013 - 14:47W 10/30
Transport of endosomes along microtubules
3 small-diameter MatTek dishes of LLCPk GFP tubulin
HBS-BSA 1mg/mL
to mix transferrin ( 3 mL) and nocodazole (15 mL) in
to rinse growth medium off before and after treatment. 6 mL per dish x 3 dishes per group x 8 groups = 144 mL. Aliquot 20 mL.
make 250 mL
Fe-HBS-BSA
to mix transferrin in
Need 2.3 mL; make 3 mL
Solution | stock conc | final conc | 1 | 2 | 2.5 | 3 | 4 | mL |
---|---|---|---|---|---|---|---|---|
Fe-citrate (mM) | 89.4 | 0.1 | 1.118 | 2.237 | 2.796 | 3.35 | 4.47 | µL |
in Fe-HBS-BSA to final volume
MAKE ENOUGH OF EACH FOR EACH GROUP TO DO 3 TREATMENTS
Transferrin in Fe-HBS-BSA
Alexa-Fluor 488 transferrin (Invitrogen T13342, MW = ~80KD)
stock is 5 mg/mL (=62.5 µM).
Final concentration = 1 µM.
- Each group may do up to 3 100 µL treatments (= 3 x 7 x 105 µL = 2.205 mL)
- Make 105 µL aliquots (21)
- Make 2.3 mL: 36.8 µL AF-488-Tf + 2.263 mL Fe-HBS-BSA
if new transferrin stock is needed add 1 mL sterile distilled water to the new bottle.
# aliquots | 1 | 2 | 3 | 6 | 9 | 12 | 15 | 18 | 21 | |
---|---|---|---|---|---|---|---|---|---|---|
vi 62.5µM tf | 1.68 | 3.36 | 5.04 | 10.1 | 15.1 | 20.2 | 25.2 | 30.2 | 35.3 | µL |
HBS-Fe-BSA | 103.3 | 206.6 | 310 | 620 | 930 | 1240 | 1550 | 1860 | 2170 | µL |
Vf 1 µM tf | 105 | 210 | 315 | 630 | 945 | 1260 | 1575 | 1890 | 2205 | µL |
Nocodazole
Acros 358240100, 10 mg
1µM in HBS-BSA
(Working concentration range is usually 100nM - 30 µM.)
Stock is 10 mg/mL (=33mM)
(Add 1mL DMSO to 10 mg in bottle = 33mM. Aliquot 3.03 µL. To make 1mM, add 97 µL.)
Make enough for each group to do two 1 mL treatments.
Aliquot 1.01 mL per tube, 15 tubes = 15 mL. Make 16 mL
- Add 97 µL HBS-BSA to 3 µL 33mM in tube to make 1 mM
- Mix 1 part 1mM with 999 parts HBS-BSA to make 1µM: 16 mL + 1.6 µL
# aliquots | 1 | 2 | 3 | 4 | 5 | 10 | 15 | |
---|---|---|---|---|---|---|---|---|
vi 1 mM noc | 1.01 | 2.02 | 3.03 | 4.04 | 5.05 | 10.1 | 15.15 | µL |
HBS-BSA | 1.009 | 2.018 | 3.027 | 4.036 | 5.045 | 10.09 | 15.135 | mL |
vf 1 µM noc | 1.01 | 2.02 | 3.03 | 4.04 | 5.05 | 10.1 | 15.15 | mL |
non-CO2 medium
to mix TMR-dextran in
to incubate cells in
TMR-dextran
in non-CO2 medium
Each group may do up to 3 treatments = 3 x 7 x 100 µL = 2.205 mL.
Invitrogen D3308, 10 mg
Make 100 mg/mL stock (add 100µL to the bottle)
1 µL aliquots
TMR-dextran Working concentration = 0.05 mg/mL in medium
100 µL per treatment
Aliquot 105 µL, make enough for each group to do 3
Make 10 mg/mL dilution to make student aliquots
1 µL 100 mg/mL stock + 9µL medium
no. aliquots | 1 | 2 | 3 | 6 | 9 | 12 | 15 | 18 | 21 | |
---|---|---|---|---|---|---|---|---|---|---|
vi (10 mg/mL TMR-dex) | 0.525 | 1.05 | 1.575 | 2.2 | 3.15 | 4.2 | 6.3 | 7.875 | 11.025 | µL |
medium | 104.5 | 128.9 | 656.7 | 875.6 | 1094.5 | 1313.4 | 1532.3 | 1751.2 | µL | |
vf (0.05 mg/mL TMR-dex) | 105 | 210 | 315 | 630 | 945 | 1260 | 1575 | 1890 | 2205 | µL |
6.1 2013
6.1 2013 kdorfman Thu, 09/26/2013 - 14:48M 11/4
Cellular responses to external stimuli
3t3 on large diameter MatTek dishes
4 dishes per group plus extra = 36
Plate 2 days before class.
Reagents 6.1 - 2013
Reagents 6.1 - 2013 kdorfman Wed, 10/30/2013 - 16:426.2 2013
6.2 2013 kdorfman Thu, 09/26/2013 - 14:49W 11/6
Release of internal calcium stores
3t3 on large-diameter MatTek dishes
4 dishes per group (plus extra) ~36
ATP
ATP kdorfman Mon, 11/04/2013 - 17:0610 µM (=5.731 mg/L) ATP in HBS
Use ATP aliquots from Lab 6.1
10 µM ATP, Calcium-free
Lab 6.2: 0.75 mL per coverslip x 2 coverslips x 8 groups = 12 mL
Make 15 mL
- 15 µL 10 mM ATP stock
- in 15 mL Ca- HBS (1 µL 10 mM ATP stock per 1 mL HBS.)
8 1.6 mL aliquots for Lab 6.2
Bradykinin for 6.2
Bradykinin for 6.2 kdorfman Mon, 11/04/2013 - 17:191mg/mL Bradykinin stock
(= 1.0602 mM) Sigma B3259 - 1 mg septum bottle
MW = 106.02
Add 1 mL H2O to the 1 mg in the bottle (NO CALCIUM! - some will be used for the calcium experiments in Lab 7.2)
2 µM Bradykinin in HBS
Use bradykinin aliquots from Lab 6.1
2 µM Bradykinin, Calcium-free
(=2.12 µg/mL)
Lab 6.2: 0.75 mL per coverslip x 2 coverslips x 8 groups = 12 mL
~2 µL 1 mg/mL stock per 1 mL working solution
31.8 µL 1 mg/mL stock in 15 mL Ca-free HBS
Make 8 aliquots of 1.6 mL
Make more if necessary. (Aliquots should have been enough for 15 mL?)
Fluo-4-AM
Fluo-4-AM kdorfman Mon, 11/04/2013 - 17:20Use aliquots from Lab 6.1
HBS for 6.2
HBS for 6.2 kdorfman Mon, 11/04/2013 - 17:21HBS needed for rinsing and to make other solutions
(3 rinses + 1 sham dilution) x 4 expts/grp x 1 mL = 16 mL
Aliquot 25 mL per group
300 mL HBS
300 mL Ca-free HBS
Ionomycin for 6.2
Ionomycin for 6.2 kdorfman Mon, 11/04/2013 - 17:24Ionomycin stock
Fisher or Invitrogen (Life Technologies) I24222
1 mM in DMSO or EtOH. 1 mg makes 1.34 mL stock.
3 µM Ionomycin in HBS
Dilute stock 1:333 in HBS: 3 µL/1 mL
24 µL in 8 mL, aliquot 800 µL
They only need 750µL, because they do one experiment in HBS, then two in Ca-free HBS.
3 µM Ionomycin in Ca-free HBS
Dilute stock 1:333 in Ca-free HBS: 3 µL/1 mL
45 µL in 15 mL
Takes a long time to thaw. Vortex.
Aliquot 1.55 mL so they can do two 750 µL experiments
Vasopressin for 6.2
Vasopressin for 6.2 kdorfman Mon, 11/04/2013 - 17:261 mg/mL (= 0.9225 mM) Vasopressin stock in water
1 µM dilution to make student solutions
1 µL 1 mg/mL in 922.5 µL water (NO CALCIUM, so stock can be used for Ca-free solutions )
10 nM (= 1.084 µg/mL) Vasopressin in HBS for students
Use Vasopressin aliquots from Lab 6.1
Calcium Free Vasopressin 10 nM
10 nM (= 1.084 µg/mL) for students
1 part 1µM dilution to 99 parts Ca-free HBS
Lab 7.2: 0.75 mL per coverslip x 2 coverslips x 8 groups = 12 mL
Make 15 mL: 150 µL + 15 mL Ca-free HBS
Make 8 aliquots of 1.6 mL
Projects 2013
Projects 2013 kdorfman Thu, 09/26/2013 - 14:50starting W 11/13
Projects
11/15/13
11/15/13 kdorfman Wed, 11/13/2013 - 20:14Super Group
- 3 MatTeks parentals 20 mm coverslip
- non-CO2 medium
11/18/13
11/18/13 kdorfman Wed, 11/13/2013 - 19:51RA & ML
- 3 MatTeks 3t3
- ATP
- Caffeine
- Fluo4-AM
Joon
- 2 coverslips 3t3
- 2 GFP-tubulin LL MatTek
- anti-tubulin
- paraglut
On the LAM
- 4 MatTeks LL GFP actin
- make serum-free for Tuesday lab
AV CW AS
- 5 coverslips LLGFP tubulin
- 1 matTek alpha tub
- non-CO2 medium
- paraglut
- anti-tubulin
Super Group
- 3 MatTeks LL parentals (heavy)
- 3 MatTek GFP tubulin (heavy)
- non-CO2 medium
- Nuc Blue
11/20/13
11/20/13 kdorfman Wed, 11/13/2013 - 19:56On the LAM
- 6 MatTek LL GFP-actin HEAVY
- no serum medium (from night before)
Dan
- 3 MatTeks LLCPk tubulin
- mitotracker
Super Group
- 6 Matteks LL tubulin HEAVY
- mitotracker
- nuc blue
RAML
- 3 Mattek LL parentals
- Fluo-4
Cool Team
- 2 mattek LL tubulin
- 2 coverslips LL tub
Joon
4 MatTek LL tubulin
11/22/13
11/22/13 kdorfman Wed, 11/20/2013 - 21:28Friday
Cool Team
LL = tubulin
- 1 coverslip
- 2 dishes
11/23/13
11/23/13 kdorfman Wed, 11/20/2013 - 21:28Saturday
Cool Team
4 dishes LL tubulin
11/25/13
11/25/13 kdorfman Wed, 11/13/2013 - 19:58Monday
RAML
6 dishes LL parentals
Joon
4 dishes LL tubulin
Cool Team
2 LL tubulin dishes
Dan
4 LL tubulin dishes
On the Lam
3 LL actin heavy dishes
Super Group
4 matteks GFP tubulin HEAVY
11/27/13
11/27/13 kdorfman Wed, 11/13/2013 - 19:59Cool Team
3 dishes, 4 coverslips tubulin
12/2/13
12/2/13 kdorfman Wed, 11/13/2013 - 20:00On the LAM
- 4 MatTeks LL GFP-actin heavy
Dan * 4 mattek llcpk GFP actin
Joon
- 4 mattek llcpk GFP tub
Cool team
- 3 mattek llcpk GFP tub *4 CVslip GFP tub
Super group
- 5 mattek GFP tub Heavy
RAML
- 4 mattek parental
12/4/13
12/4/13 kdorfman Wed, 11/13/2013 - 20:1412/6/13
12/6/13 kdorfman Wed, 12/04/2013 - 21:41Super Group
4 heavy LL tubulin MatTeks
Cool Team
2 MatTek LL tub
1 coverslip LL tub
12/8/13
12/8/13 kdorfman Thu, 12/05/2013 - 15:18RAML
4 LL parentals, in dishes
4 3t3, in dishes
Microscopes
Microscopes kdorfman Mon, 01/09/2012 - 15:20Nikon Filter Cubes
label on filter wheel | excitation wavelength | emission wavelength | used for | label on cube |
---|---|---|---|---|
UV | UV (360 nm) | blue (460 nm) | DAPI | UV-2E-C |
B | B (480 nm) | green (535 nm) | GFP, fluorescein | B-2E/C |
G | G ( 560 nm) | red (630 nm) | Texas red, rhodamine | Y-2E/C |
LP | B (470 nm) | long pass green - red (>500 nm) | chlorophyll & GFP simultaneously | B-2A |
mOrange1 | G (530 nm) | orange (575). | m orange | Chroma 49014 |
Details:
Ultraviolet Excitation Filter Block UV-2E/C Specifications:
Excitation Filter Wavelengths: 340-380 nanometers (bandpass, 360 CWL)
Dichromatic Mirror Cut-on Wavelength: 400 nanometers (longpass, LP)
Barrier Filter Wavelengths: 435-485 nanometers (bandpass, 460 CWL)
Blue Excitation Filter Block B-2E/C Specifications:
Excitation Filter Wavelengths: 465-495 nanometers (bandpass, 480 CWL)
Dichromatic Mirror Cut-on Wavelength: 505 nanometers (longpass, LP)
Barrier Filter Wavelengths: 515-555 nanometers (bandpass, 535 CWL)
Yellow Excitation Filter Block Y-2E/C Specifications:
Excitation Filter Wavelengths: 540-580 nanometers (bandpass, 560 CWL)
Dichromatic Mirror Cut-on Wavelength: 595 nanometers (longpass, LP)
Barrier Filter Wavelengths: 600-660 nanometers (bandpass, 630 CWL)
Blue Excitation Filter Block B-2A Specifications:
Excitation Filter Wavelengths: 450-490 nanometers (bandpass, 470 CWL)
Dichromatic Mirror Cut-on Wavelength: 500 nanometers (longpass, LP)
Barrier Filter Wavelengths: 515 nanometer cut-on (longpass, LP)
mKO/mOrange Specifications:
Excitation Filter Wavelengths: 515-545 nm
Dichromatic Mirror cut-on wavelength: 550 nm
Barrier Filter Wavelengths: 555 - 595 nm
NIkon repairs (every summer)
contact John DeToma: johnd@mvi-inc.com
They need a PO before they schedule an on-site service appointment
-
installed on scope 7 for Akiko Okusu's class ↩︎
Aligning the xenon arc lamp
Aligning the xenon arc lamp kdorfman Wed, 04/10/2019 - 18:34DIC
DIC bcrcstaff Tue, 11/20/2018 - 15:29(On microscope 7)
Prism combinations
objective | magnification | prism on condenser turret | prism on objective turret |
---|---|---|---|
Plan Fluor phase | 10x | N1 | 10x |
Plan Fluor phase | 40x (short working distance) | N2 | 40x I |
S Plan Fluor phase | 40x (extra-long WD) | N2 | 40x III |
Plan Fluor phase | 100x oil | N2 | 100x II |
Achromat w/iris brightfield | 100x oil | no DIC |
microscope pictures
microscope pictures kdorfman Fri, 07/20/2018 - 14:20Posters
Posters kdorfman Wed, 12/03/2014 - 18:42Supplies
Supplies kdorfman Thu, 11/03/2011 - 19:35Get coverslip-bottom dishes from Cellvis, much cheaper than MatTek.
Celltreat also has them, about $2/dish:
https://www.celltreat.com/30mm-x-10mm-tissue-culture-treated-dish-15mm-…
Microscope bulbs for phase: 12V 100 W
- Philips 77241 or
- Osram HLX 64623
http://www.planetbulb.com/osram-eva-64623-hlx-54251/
Microscope bulbs for Zeiss:
- 38-01-77 6V 15W
- OQ-77Z 3800-18-1740 6V 15W or
- WW-6W51-8 from interlight.biz
Microscope bulbs for Olympus: BLC 120V 30W S11 BA15D from interlight.biz
Nucleofection kit
Ingenio Electroporation kit, MIR50112
mounting medium (SouthernBiotech Dapi Fluoromount-G, Fisher OB010020)
Hoechst solution Invitrogen™ Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg/mL Solution in Water Nucleic acid stain Invitrogen™ H3570 cheaper than Thermo Fisher
Immersion Oil
For upright microscopes: Low viscosity
For inverted: high viscosity
Roy Kinoshita (MVI) says to use Type B oil ($10.00 for 1oz) for the fluorescence scopes.
Nikon recommends Type B for their Perfect Focus System (PFS), which maintains focus continuously on the inverted, almost always in fluorescence, and designed for long-term imaging. If the oil dries up or seeps down from low viscosity, PFS would fail after a few hours of imaging and people would not be happy. I believe the viscosity is 1250 cs.
From Cargille:
type | viscosity | RI @ 546 | background fluorescence |
---|---|---|---|
A | 150 | 1.518 | low |
FF | 170 | 1.4811 | virtually zero |
LDF | 500 | 1.518 | very very low (for high res fluorescence) |
HF | 700 | 1.518 | very very low |
37LDF | 1250 | 1.5181 | low (for use only at 37C) |
B | 1250 | 2100 | low |
NVH | 21000 | 1.5180 | low (larger coverslip-to-lens distance) |
OVH | 46000 | 1.5178 | low |
!2023 Wadsworth BioImaging
!2023 Wadsworth BioImaging kdorfman Fri, 01/13/2023 - 17:39Spring 2023
Handouts saved in OneDrive here
2023 Project 1
2023 Project 1 kdorfman Wed, 03/29/2023 - 14:33W 3/29/23
Treatment | Stock concentration | Dilute in |
---|---|---|
Latrunculin | 1 mM | serum-free medium (e.g., Fluorobrite) |
Taxol | 10 mM | medium |
Nocodazole | 33 mM | DMSO to 1 mM, medium to 3.3 uM |
STLC | 1 mM | medium |
GSK-923295 | ||
MG132 |
Fixatives:
Media:
Stains:
2023 Project 1 day 2
2023 Project 1 day 2 kdorfman Wed, 03/29/2023 - 21:31For M 4/3/23
Grp | dish or coverslip | strain | reagents |
---|---|---|---|
1 | 3 dishes | GFP tubulin | GSK, |
2 | 3 coverslips | GFP tubulin | GSK, fix & DAPI |
3 | 4 dishes | GFP tubulin | Taxol, nuc blue |
4 | 6 coverslips | GFP tubulin | Latrunculin, serum-free medium, phalloidin, fix & DAPI |
5 | 4 dishes | GFP | STLC, DMSO, nuc blue |
6 | 6 coverslips | parental | MG 132, fix, anti alpha tubulin, goat anti-rat-FITC, DAPI |
7 | 3 coverslips | GFP | Taxol, fix & DAPI |
8 | 6 coverslips | GFP | MG132, methanol, hec 1 & DAPI |
2023 Project 1 day 3
2023 Project 1 day 3 kdorfman Mon, 04/03/2023 - 20:57W April 5
Group | Cells | dish or coverslip | reagents |
---|---|---|---|
1 | GFP | 3 coverslips | |
2 | nothing | all set | |
3 | GFP | 6 dishes | Taxol, nuc blue |
4 | GFP tubulin | 6 coverslips | Latrunculin, serum-free medium, phalloidin, fix & DAPI |
5 | GFP | 2 dishes | |
6 | no cells | all set | anti alpha tubulin, goat anti-rat-FITC, DAPI |
7 | GFP | 3 coverslips | |
8 | GFP | 6 coverslips |
2023 Project 1 day 4
2023 Project 1 day 4 kdorfman Wed, 04/05/2023 - 20:26for Monday 4/10/2023
Group | dishes | coverslips | cell type | reagents |
---|---|---|---|---|
1 | 1 | GFP-tub | GSK 923295 | |
2 | ||||
3 | ||||
4 | ||||
5 | 3 | GFP-tub | STLC | |
6 | ||||
7 | ||||
8 |
2023 Project 2
2023 Project 2 kdorfman Tue, 04/11/2023 - 17:312023-May
2023-May kdorfman Tue, 05/02/2023 - 15:05Started | Needed | cells1 | 12.52 | 25 | 75 | dish3 | slips4 | treated | for |
---|---|---|---|---|---|---|---|---|---|
M 5/1 | W 5/3 | LL | 1 | 2 | 1 | ||||
LL | 4 | 300 nM centrinone | J | ||||||
HeLaC | 2 | 250 nM centrinone | J | ||||||
W 5/3 | F 5/5 | LL | 1 | 1 | C: large numbers | ||||
F 5/5 | S, S | LL | |||||||
M 5/8 | LL | ||||||||
HeLaC | |||||||||
M 5/8 | W 5/10 | LL | |||||||
HeLa | |||||||||
HeLaC |