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        Media and Solutions Required for Routine ES Cell Culture 1

        互联网

        1173

         

        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
        - 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

         

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