MEDIA AND SOLUTIONS REQUIRED FOR ROUTINE ES CELL CULTURE

 

Media Used

To prepare 100 ml medium

DMEM	80 ml
FCS	15 ml
Non-essential amino acids (100x)	1 ml
Pen/strep (5,000 1U/ml, 5000 ug/ml)	1 ml
L-Glutamine 200 mM	1 ml
Nucleosides stock (100x)	1 ml
BME 0.1M	0.2 ml

Dulbecco's Modification of Eagles Medium (DMEM)

1 x without L-glutamine with 4.5 g/L Glucose. Store at 4�

Cytosystems 500 ml Cat. No. 11.016.0500V

 

Non essential amino acids 100x

Cytosystems 100 ml Cat No. 21-145-0100V. Store at 4�

 

Penicillin - streptomycin (5,000 1U/ml, 5,000 ug/ml)

Cytosystems 100 ml Cat No. 21-140-0100V. Store at -20�

 

Foetal Calf Serum (FCS)

Needs to be tested for ability to support growth of ES Cells. Serum should be stored frozen and heat inactivated before use by heating at 56� for 30 mins. It may be stored at 4� following inactiavation. The heat inactivation removes complement activity which along with natural heterophile antibodies, may be toxic for ES cells.

 

100 x Nucleoside Stock

To prepare 100 ml (100x)

Adenosine	80 mg	3mM	Sigma A 4036
Guanosine	85 mg	3mM	Sigma G 6264
Cytidine	73 mg	3mM	Sigma C 4654
Uridine	73 mg	3mM	Sigma U 3003
Thymidine	24 mg	1mM	Sigma T 1895

Add to 100 ml Travenol water and dissolve by warming to 37�. Filter sterilize and aliquot while warm. Store at 4� for months. The nucleosides will come out of solution. Warm to 37� before use to re-solubilize. Solution seems to be stable at 4 C.

Other solutions required.

- 1 x Dulbecco's Phosphate Buffered Salt Solution (PBS) without calcium and magnesium.

	Cytosystems	100 ml	11.075.0100V
		500 ml	11.075.0500V

- Trypsin 2.5% Cytosystems 100 ml 21-159-0100V

- Trypsin - EDTA (1:250) Cytosystems (0.05% Trypsin) 100 ml 21-160-0100V

Solutions to be made up

PBS/EGTA (0.5 mM)

To 100 mls of PBS add 1 ml of 0.05M stock EGTA (Sigma E4378) to give a final concentration of 0.5 mM.

 

Stock EGTA in H20 0.05M add conc. NaOH to dissolve

 

Trypsin 0.25%.

To 20 mls Cytosystems Trypsin/EDTA 0.05%, add 1.6 mls 2.5% Trypsin

 

0.1% Gelatin in PBS

To 100 mls PBS add 0.1 gm Gelatin Sigma G-1890 and autoclave

 

0.1M 2 - mercaptoethanol (�E) Sigma M-6250

Add 0.1 ml �E (14.4M) to 14.3 mls PBS and filter through a 0.2 uM ACRODISC. Store at -20� for up to one month.

 

LIF (if no feeders are used)

LIF ESGRO AMRAD (murine Lif in PBS/BSA solution) 1 ml ampoule

107 U/ml. Dilute 1/100 in DMEM 10% FCS, aliquot into 10 x 10 ml tubes,

store at -20� until use.

107 U/ml dilute 1/100 = 105 U/ml. (100 x conc)

Dilute 1/100 for use = 1000 U/ml