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 - 19:20| 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
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 - 19:48| 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)
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 - 20:05| 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
cells Sat 12/1 kdorfman Wed, 11/28/2012 - 20:16| 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
cells Wed 12/5 kdorfman Mon, 12/03/2012 - 16:36| 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 |