Tina:Brainbow induction with Cre recombinase (BBI)

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Revision as of 01:40, 17 July 2012 by >TinaLo (Created page with "==Osteogenic differentiation media composition test== *'''Purpose:''' To test the effects of osteogenic differentiation media on mESCs *'''Test #1:''' **Date: 7/1/2012 ~ ongoi...")
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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.