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°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)
| 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°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.| Cytosystems | 100 ml | 11.075.0100V | | Cytosystems | 500 ml | 11.075.0500V |
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| - | 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. |
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| - | 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 |
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| - | 0.1% Gelatin in PBS| - | To 100 mls PBS add 0.1 gm Gelatin Sigma G-1890 and autoclave |
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| - | 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. |
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| - | 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 |
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