Tina:Brainbow induction with Cre recombinase (BBI)
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Osteogenic differentiation media composition test
- Purpose: To test the effects of osteogenic differentiation media on mESCs
- Test #1:
- Date: 7/1/2012 ~ ongoing work
- Cells: P10 InsCAGGsAlov3 (mES brainbow cells)
- Osteogenic differentiation media (with 1% serum):
Component'' | Volumn | Final concentration |
MEM | 193ml | |
Serum | 2ml | 1% |
Dexamethasone | 200ul of 0.1mM stock | 0.1uM |
Ascorbic acid | 1ml of 10mg/ml stock | 50ug/ml |
b-GP | 2ml of 1M stock | 10mM |
PenStrep | 2ml | 1% |
Total | 200 mL |
- 7/1: P9 mES brainbow cells (on feeder) were trpsinized, let sit for 10 min, and then seeded 1:5 (two wells) and 1:10 (4 wells) onto feeder free system (now become P10).
- 7/2 and 7/3: Cell density was still low. Induction should wait for one more day.
- 7/4: Cell density became too high. Several colonies were formed. Therefore, the best time point to induce osteogenic differentiation is ‘’1-2 days after seeding’’’ (need further confirmation).
- Add induction media to each well on 7/4 to observe the cell morphology changes at the beginning of osteogenic differentiation.
- 7/5: Observe lots of cell death within each well. The concentration of chemicals in induction media might need further adjustment. (Pictures.)
Seeding density test
- Purpose: To determine the seeding density of mESCs for osteogenic differentiation
- Test #1:
- Date: 7/5/2012 ~ ongoing work
- Cells: P11 InsCAGGsAlov3 (mES brainbow cells)
- 7/5: P10 mES brainbow cells (on feeder) were trypsinized. Cells were then divided into two groups. Group one were seeded 1:2, 1:4, 1:6, 1:8 and 1:12 on gelatin coated glass-well immediately. Group two were let sit for 15 min and then seeded 1:2, 1:4, and 1:6 on gelatin coated glass-well.
- 7/6 (next day of seeding): Take pictures of each well. Find out the best seeding manner and seeding density.