Human Embryonic Stem (ES) Cell Protocols――Media and Reagents
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Serum Free Media for human ES cells on MEFs: can last for 7-10 days
| Final Concentration |
Amount for 250ml Stock solution
|
|---|---|
| 80% DMEM-F12 |
200ml
|
| 20% KO Serum Replacer |
50ml
|
| 1% Non-essential Amino Acids |
2.5ml
|
| 1mM L-glutamine (see recipe) |
2.5ml
|
| 0.1mM b-mercaptoethanol |
part of L-glutamine
|
| 4ng/ml bFGF (see recipe) |
0.5ml
|
L-glutamine stock solution: Prepare on a per-use basis
- 0.146g L-glutamine
- 10ml Ca/Mg free PBS
- 7 ul b-mercaptoethanol
bFGF Stock: Store aliquots at �20 or �70
- 10ug vial of bFGF
- 5ml 0.1% Fraction V BSA
- Aliquot 0.5ml into sterile tubes
0.1% Fraction V BSA:
- 0.1g Fraction V BSA
- 100ml PBS with Ca and Mg
- Filter Sterilize
Collagenase IV Split Media: This media lasts 2-3 weeks
- Final concentration of 1mg/ml in DMEM-F12 media ie. 0.05g collagenase IV in 50ml DMEM-F12
Cell Freezing Media:
| Final Concentration |
Amount for 10ml Stock Solution
|
|---|---|
| 60% DMEM-F12 |
6ml
|
| 20% defined FBS |
2ml
|
| 20% DMSO (do not filter sterilize) |
2ml
|
| Final Concentration |
Amount for 10ml Stock Solution
|
|---|---|
| 80% DMEM-F12 |
8ml
|
| 20% defined FBS |
2ml
|
