Media Used

To prepare 100 ml medium

DMEM80 ml
FCS15 ml
Non-essential amino acids (100x)1 ml
Pen/strep (5,000 1U/ml, 5000 ug/ml)1 ml
L-Glutamine 200 mM1 ml
Nucleosides stock (100x)1 ml
BME 0.1M0.2 ml

Dulbecco's Modification of Eagles Medium (DMEM)

1 x without L-glutamine with 4.5 g/L Glucose. Store at 4°C
Cytosystems 500 ml Cat. No. 11.016.0500V

Non essential amino acids 100x

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

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

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

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°C for 30 mins. It may be stored at 4°C following inactivation. 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)
Adenosine80 mg3mMSigma A - 4036
Guanosine85 mg3mMSigma G - 6264
Cytidine73 mg3mMSigma C - 4654
Uridine73 mg3mMSigma U - 3003
Thymidine24 mg1mMSigma T - 1895
Add to 100 ml Travenol water and dissolve by warming to 37°C. Filter sterilize and aliquot while warm. Store at 4°C for months. The nucleosides will come out of solution. Warm to 37°C 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.
Cytosystems100 ml11.075.0100V
Cytosystems500 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 (ßME) Sigma M-6250
-Add 0.1 ml ßME (14.4M) to 14.3 mls PBS and filter through a 0.2 uM ACRODISC. Store at -20°C 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°C until use.
-107 U/ml dilute 1/100 = 105 U/ml. (100 x conc)
-Dilute 1/100 for use = 1000 U/ml