Bioimaging Instructor

Here are the preparation procedures for Bioimaging labs.

Bioimage Calendar

Google calendars

isbbiology at gmail dot com

Wahoo2012

Bioimaging calendar

0: Pre-sem prep

Pre-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

www.mirusbio.com

Fisher: MIR50112 $230.17. More expensive than mirusbio, but mirus charges $35 for shipping.

Solutions

Solutions 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

Students use lots of these:

  • 10 mL PBS
  • 10 mL serum-free-non-CO2 medium
  • 10 mL HBSS
  • 10 mL HBS

Slides

slides 2012

CHECK 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

Lab 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

LL'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

Alpha actinin

Stains 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

Stains Centrosomes

  • Monoclonal, GTU-88, Sigma,
  • Fixation: methanol; para/glut
  • Dilution 1:100

Golgi 58K

  • Sigma G2404, mouse monoclonal
  • Formaldehyde (no glut)
  • 1:100

Hec1

Stains kinetochores

  • Novus biological, recombinant human HEc1, monoclonal
  • Fix in methanol
  • 1:200

LAP2

Stains nuclear envelope

  • Methanol
  • 1:100
  • BD transduction
  • Mouse IgG

Vinculin

Focal adhesion plaques

  • Monoclonal, sigma V4505
  • Formaldehyde
  • 1:100

2016 schedule

Lab 1 1 DAPI slide per pair

Lab 2

Lab 3 Numerical aperture . same DAPI slide

Lab 4 Resolution.

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

Lab 8 - Organelles 10/3/16

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

Lab 10 10/12

Lab 11.1 Motility I 10/17

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

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Cell culture

CO2 Incubator

  • 37C
  • 5% CO2
  • pan of water in bottom

Freezing

Approximately 1 freezing vial per 25 cm2 flask.

3 vials from large size 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 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
  • Put into liquid nitrogen.

Plating

  • 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

  • Put 5 mL medium in each flask. Label with cell type and passage number
  • Warm flasks in incubator
  • Remove medium from cells.
  • Rinse once with ~3 mL sterile PBS. (Squirt in, then suck out.)
  • Add 15 drops sterile trypsin (=~0.75 mL)
  • Incubate (37C) 5 min
  • Check to make sure you can see them floating in the liquid
  • Add ~1mL medium to flask, draw up and down to mix.
  • Put
    • ~4-8 drops (= 0.2 – 0.4 mL) into each new flask containing medium
    • ~4-5 drops ( = 0.2 – 0.25 mL) if cells are 70 – 80% confluent
  • Put back in incubator.

Thawing

  • 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

1.1: intro to microscopes

1.2: image formation

2.1: Numerical aperture

need 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

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2.2: Resolution

3.1: Fluorescence

Fluorescence 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

Immunofluorescence 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

Immunofluorescence Labeling

3.3: Direct fluor

Direct 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

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3.4 GFP-tags

Live 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

Lipofectamine2000 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

Nucleofection

Nucleofection 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

Plasmids

gene fluorophore antibiotic 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

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

4.1: Cell cycle

Cell 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

Motility

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

CHECK 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

Receptor-mediated Endocytosis

Monday 10/17/11

Materials for 6.1

  • 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 on coverslips

For lab on Monday 10/16/11

Each group gets 4 coverslips of a given type of cell, either 3t3 or LLCPk.

On Friday 10/14/11: Prepare 32 tiny petri dishes, each with 1 sterile coverslip.

On Saturday 10/15/11: Plate:

  • 16 coverslips 3t3
  • 16 coverslips LLCPk

Reagents for 6.1

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 for 6.1

Cells for 6.1

3t3 on coverslips

3 dishes per group, plus extras = 24

Plate 2 days before lab.

Reagents 2012

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
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

Bulk-phase Endocytosis

Cells for 6.2 2012

3t3

2 coverslips per group

2 small diameter MatTeks per group

Cells for 6.2

Lab 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

Monday 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

Transport of Endosomes along Microtubules

Wednesday 10/26/11

Cells for 6.3

LLCPk-GFP-tub

3 small diameter MatTek dishes per group

Plate 24 dishes 2 days before lab

Reagents for 6.3

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 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

Release of Internal Calcium Stores

Wednesday, 11/2/11

Rescheduled for Monday, 11/7/11, due to snowday 10/31/11

7.1: External stimuli

Cellular Responses to External Stimuli
Monday, 10/31/11

Rescheduled for 11/2/11 because of storm on 10/31/11

Cells for 7.1

3t3 on large diameter MatTek dishes

4 dishes per group plus extra = 32

Plate 2 days before class.

Reagents for 7.1

HBS

HBS for washes and making solutions

25 mL per group

(total = 175 mL)

Make from 10X

Make 350 mL

Pluronic F-127

Stock

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

10 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

1 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 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

1 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

This page tests the use of collapsible sections for recipe pages

Cells for 7.2

3t3 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

Use left-over aliquots of stimulants from 7.1 with calcium, make calcium-free reagents for 7.2

ATP

10 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

1mg/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

Use Fluo-4 aliquots from Lab 7.1

HBS

HBS 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 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

1 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

Projects

Have students make a class poster for the bulletin board in the hall.

Chi-Square calculator

from http://www.quantpsy.org/chisq/chisq.htm

ER-tracker

ER-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

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HBSS/Ca/Mg

Hank's Balanced Salt Solution

Gibco cat. #14025-092

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

Alexis 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

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Projects 2012

For 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 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

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Cells Monday 11/26

Names 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

For 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

Names 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)

Sigma 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

Names 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

Names 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

Names 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

Cells for 11/28/11

Group 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

Group 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

Group 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

Group 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

1.1 2013

W 9/04

Intro to Equipment

1.2 2013

W 9/09

Imaging mitosis with fixed cells. Image Spatial Quantification

1.3 2013

W 9/11

Image Formation in the Microscope

2.1 2013

M 9/16

Numerical Aperture

2.2 2013

W 9/18

Resolution

3.1 2013

M 9/23

Fluorescence Microscopy

3.2 2013

W 9/25

Identification of Cellular Organelles

3.3 2013

M 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

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

W 10/2

Imaging Living Cells Expressing GFP-Tagged Proteins

2014

  • LL-EB1-GFP stable line

  • LL-alpha tubulin=GFP stable line

** transfected**

  • H2B m cherry

  • alpha actininin (GFP?)

4.1 2013

W 10/9

Motility in Control Cells

On MatTek dishes:

  • 3t3

  • B16

  • LL-GFP actin

Non-CO2 medium

4.2 2013

Tu 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.

5.1 2013

W 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

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

M 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

Cells

  • 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

HBS-BSA

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

Cells

  • 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

W 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

To make mL HBS-BSA
add: mL 10X HBS
add: g BSA
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


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

M 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.

6.2 2013

W 11/6

Release of internal calcium stores

3t3 on large-diameter MatTek dishes

4 dishes per group (plus extra) ~36

ATP

10 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

1mg/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

Use aliquots from Lab 6.1

HBS for 6.2

HBS 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 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

1 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

starting W 11/13

Projects

11/15/13

Super Group

  • 3 MatTeks parentals 20 mm coverslip
  • non-CO2 medium

11/18/13

RA & 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

On 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

Friday

Cool Team

LL = tubulin

  • 1 coverslip
  • 2 dishes

11/23/13

Saturday

Cool Team

4 dishes LL tubulin

11/25/13

Monday

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

Cool Team

3 dishes, 4 coverslips tubulin

12/2/13

On 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

12/6/13

Super Group

4 heavy LL tubulin MatTeks


Cool Team

2 MatTek LL tub

1 coverslip LL tub

12/8/13

RAML

4 LL parentals, in dishes

4 3t3, in dishes

Microscopes

Nikon Filter Cubes

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)
AttachmentSize
image_spatial_quantification.pdf188.27 KB
image_spatial_quantification.docx289.88 KB
lab1-2016bioimage-manual-final.pdf412.17 KB
pages_59_477h-2015-manual-2.pdf110.63 KB

Posters

AttachmentSize
bioimaging_s09.ppt1.32 MB
bioimaging_poster_2008_kd.ppt13.25 MB

Summer projects

Supplies

Get coverslip-bottom 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

Celltreat also has them, about $2/dish:

https://www.celltreat.com/30mm-x-10mm-tissue-culture-treated-dish-15mm-g...


Microscope bulbs for phase: 12V 100 W

http://www.planetbulb.com/osram-eva-64623-hlx-54251/

Microscope bulbs for Zeiss:

Microscope bulbs for Olympus: BLC 120V 30W S11 BA15D from interlight.biz


Nucleofection kit

Ingenio Electroporation kit, MIR50112

www.mirusbio.com


mounting medium (SouthernBiotech Dapi Fluoromount-G, Fisher OB010020)