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To prepare 100 ml medium
|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 4ĄC
Cytosystems 500 ml Cat. No. 11.016.0500V
Non essential amino acids 100x
Cytosystems 100 ml Cat No. 21-145-0100V. Store at 4ĄC
Penicillin - streptomycin (5,000 1U/ml, 5,000 ug/ml)
Cytosystems 100 ml Cat No. 21-140-0100V. Store at -20ĄC
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 56ĄC for 30 mins. It may be stored at 4ĄC 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 37ĄC. Filter sterilize and aliquot while warm. Store at 4ĄC for months. The nucleosides will come out of solution. Warm to 37ĄC 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.
- 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
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 (§ME) Sigma M-6250
Add 0.1 ml §ME (14.4M) to 14.3 mls PBS and filter through a 0.2 uM ACRODISC. Store at -20ĄC 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 -20ĄC until use.
107 U/ml dilute 1/100 = 105 U/ml. (100 x conc)
Dilute 1/100 for use = 1000 U/ml
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