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RESEARCH DIVISION Laboratory Manual

 


 

Media and Solutions Required for Routine ES Cell Culture (Version 1)

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 4C
Cytosystems 500 ml Cat. No. 11.016.0500V

Non essential amino acids 100x

Cytosystems 100 ml Cat No. 21-145-0100V. Store at 4C

Penicillin - streptomycin (5,000 1U/ml, 5,000 ug/ml)

Cytosystems 100 ml Cat No. 21-140-0100V. Store at -20C

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 56C for 30 mins. It may be stored at 4C 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 37C. Filter sterilize and aliquot while warm. Store at 4C for months. The nucleosides will come out of solution. Warm to 37C 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 (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 -20C 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 -20C until use.
- 107 U/ml dilute 1/100 = 105 U/ml. (100 x conc)
- Dilute 1/100 for use = 1000 U/ml
Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail: d.bowtell@pmci.unimelb.edu.au
David Bowtell PMCI October 1998