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  • sect50
  • MEDIA AND SOLUTIONS REQUIRED FOR ROUTINE ES CELL CULTURE

    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 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)

    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 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.

    Cytosystems100 ml11.075.0100V
    500 ml11.075.0500V

    - 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 (§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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    This page is maintained by David Bowtell (bowtell@ariel.ucs.unimelb.edu.au) using HTML Author. Last modified on 10/24/95.