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GGTC German Gene Trap Consortium

 

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 media

 

 

1. ES - cell culture media and solutions

2. MEF (mouse embryonic fibroblast) medium (= feeder cell medium)

3. tbv - 2 ES medium

 

4. F1 ES medium

5. R1 ES medium

6. CJ 7 ES medium

7. E14Tg2A medium

 

 

download the media pdf-file here

 

1. ES-cell culture media and solutions
(kindly provided by Susanne Bourier, Ernst-Martin Füchtbauer, Ralf Kühn and Melanie Hollatz)

 

 

Materials

  • gelatinized plates (NUNC)
  • Gelatine solution: 0.1 % (Sigma cat. # G1393) in aq (Fresenius)
  • PBS (Dulbecco´s phosphate-buffered saline without Mg2+ and Ca2+; Gibco cat. # 14190-094)
  • Trypsin-EDTA (1x) in HBSS without Mg2+ and Ca2+ (Gibco cat. # 25300-054)
  • Dulbeccos´ Modified Eagles Medium (DMEM + 4500 mg/L Glucose + L-Glutamine + Pyruvate; Gibco cat. # 41966-029)
  • Glasgow´s Modified Eagles Medium (BHK-21 + L-Glutamine – Tryptose Phosphate Broth; Gibco cat. # 21710-025)
  • Geneticin (G418 Sulfate), e.g. Gibco cat. #10131-027; stock 200 mg/ml (4°C)
  • DMSO (Dimethyl sulfoxide; Sigma cat. # D5879)
  • 2x conc. freezing medium ES cells: 50% FCS, 20% DMSO. Make fresh before use.
  • L-Glutamine 200 mM (100x) (Gibco cat. # 25030-032)
  • Fetal Calf Serum (PAN Biotech, cat. # 2602-P242901)
  • Hepes (1M) (Gibco cat. # 15630-056)
  • MEM nonessential Amino Acids (100x) (Gibco cat. # 11140-035)
  • MEM Sodium Pyruvate 100 mM (Gibco cat. # 11360-039)
  • 2- Mercaptoethanol 50 MM (Gibco cat. # 31350-010), use up within 2 weeks or freeze stocks.
  • Penicillin/Streptomycin (Gibco cat. # 15140-122); aliquots at -20°C
  • 10 7 units/1ml LIF (leukocyte inhibitory factor, Chemicon cat. # ESG1107)
  • nucleosides 100ml 100x stocksolution (for CJ7 cells):
    80 mg Adenosine (Sigma A-4036)
    73 mg Cytidine (Sigma C-4654)
    85 mg Guanosine (Sigma G-6264)
    24 mg Thymidine (Sigma T-1895)
    73 mg Uridine (Sigma U-3003)

    dissolve in 100 ml H2O (pre-warm to 37°C) and filter-sterilize. Aliquots (e. g. 5 ml Bluecap) can be stored at 4°C. Precipitate redissolves at 37°C.

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2. MEF (mouse embryonic fibroblast) medium (= feeder cell medium)

 

Dulbeccos´ Modified Eagles Medium, high glucose, with Na-Pyruvate containing 10% (v/v) FCS Fetal Calf Serum, 2 mM Glutamine, 100x nonessential Amino Acids, 10- 4 M ß-Mercaptoethanol,

optional: 50 U/ml Pen/Strep.

 

3. tbv-2 ES medium

 

Dulbeccos´ Modified Eagles Medium, high glucose, with Na-Pyruvate containing 15% (v/v) FCS Fetal Calf Serum, 2 mM Glutamine, 100x nonessential Amino Acids, 10- 4 M ß-Mercaptoethanol; Add before use: 1000 U/ml leukocyte inhibitory factor (LIF),

optional: 50 U/ml Pen/Strep.

For selection add 200 ug/ml G418 to each DMEM bottle.

 

4. F1 ES medium

 

Dulbeccos´ Modified Eagles Medium, high glucose, with Na-Pyruvate containing 15% (v/v) FCS Fetal Calf Serum, 2 mM Glutamine, 100x nonessential Amino Acids, 1M Hepes, 10- 4 M ß-Mercaptoethanol; Add before use: 1000 U/ml leukocyte inhibitory factor (LIF),

optional: 50 U/ml Pen/Strep.

 

5. R1 ES medium (A. Nagy et al.(1993): Proc Natl Acad Sci U S A. Sep 15;90 (18): 8424 – 8.)

 

Dulbeccos´ Modified Eagles Medium, high glucose, without Na-Pyruvate containing:

20% (v/v) FCS Fetal Calf Serum, 1 mM Na-Pyruvate, 2 mM Glutamine, 10- 4 M ß-Mercaptoethanol, 5.7 ml nonessential Amino Acids (from 100x stock); Add before use: 1000 U/ml leukocyte inhibitory factor (LIF);

optional: 50 U/ml Pen/Strep.

 

6. CJ7 ES medium

Dulbeccos´ Modified Eagles Medium, high glucose, without Na-Pyruvate containing:

15% (v/v) FCS Fetal Calf Serum, 2 mM Glutamine, 10- 4 M ß-Mercaptoethanol, 5.7 ml nonessential Amino Acids (from 100x stock), 5 ml nucleoside (100x), Add before use: 1000 U/ml leukocyte inhibitory factor (LIF);

optional: 50 U/ml Pen/Strep.

 

7. E14Tg2A medium (according to Bay Genomics)

ES cell medium: 1x GMEM medium (Sigma, G5154) supplemented with 2 mM glutamine (Gibco-BRL), 1 mM sodium pyruvate (Gibco-BRL), 1x nonessential amino acids, 10% (v/v) fetal bovine serum (characterized, Hyclone), a 1:1000 dilution of beta-mercaptoethanol stock solution, and 500-1000 units per ml of leukocyte inhibitory factor (Chemicon).

Trypsin solution: Add 100 mg of EDTA tetrasodium salt (Sigma) to 500 ml of PBS. Filter sterlize and add 10 ml (for 1x) or 20 ml (for 2x) of 2.5% trypsin solution (Gibco-BRL) and 5 ml of chicken serum (Gibco-BRL). Store in 20 ml aliquots at -20°C. (Note: 2.5% trypsin solution should be aliquoted and stored at -20°C to avoid multiple freeze-thawing cycles.)

Geneticin (Gibco-BRL): Dissolve powder in PBS to make a 125 mg/ml stock solution (active concentration). Filter sterilize and store at -20°C. Add 0.56 ml to each bottle (560 ml) of ES cell medium. (Note: The concentration of Geneticin should be titrated to determine the minimum concentration that will kill nontransfected ES cells in 5 days.)

 

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