Dec. 18, 1990
R. Veile
To 1 liter of sterile RPMI 1640 with 2mM L-glutamine, add:
165.0 ml fetal bovine serum, heat inactivated
12.0 ml 200mM (100X) L-glutamine
12.0 ml antimycotic/antibiotic (100X), liquid, Gibco, Cat. No. 600-5240AG
Filter sterilize through a 0.22 祄 cellulose acetate filter and store up to 2 weeks at 4 degrees C.
1X Cyclosporin media: (100 ml)
To 100 ml of growth media add:
1.0 ml 100X Cyclosporin A
2X Cyclosporin A media: (1 礸/ml)
To 100 ml of growth media add 2 ml of 100X Cyclosporin A.
100X Cyclosporin A: (100ml)
Dissolve 1 mg CSA in 0.1 ml ethanol in a sterile 15 ml centrifuge tube with a small magnetic stirrer. Add 0.02 ml (= 20 祃 ) of Tween 80 and mix well. While continually stirring, add 1 ml RPMI drop by drop. Bring to a final volume of 100 ml with RPMI. Filter sterilize with a 0.22 祄 filter. Store at 4 degrees C for up to 4 months.
Freezing media: (1 liter)
Prepare a 1 liter volume and divide into 25-50 ml centrifuge tubes containing 40 ml each.
Store the tubes at -80 degrees C for up to one year.
700 ml RPMI-1640 with 2mM L-Glutamine
200 ml fetal bovine serum (FBS)
100 ml dimethyl sulfoxide (DMSO, Sigma)
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1000 ml total volume
Note: Filter sterilize media and FBS with a 0.22 祄 cellulose acetate filter. Do NOT filter DMSO, it will dissolve the cellulose acetate membrane.
Growth media: (600 ml)
To 500 ml of sterile RPMI 1640 with 2 mM L-glutamine, add:
90.0 ml FBS
6.0 ml 200 mM (100X) L-glutamine
0.6 ml 50 mg/ml gentamicin reagent
Note: Filter sterilize through a 0.22 祄 filter and store up to 2 weeks at 4 degrees C.
Growth media: (1 liter)
To 1 liter of strerile RPMI 1640 with 2mM L-glutamine, add:
165.0 ml fetal bovine serum, heat inactivated at 50-60 degrees C for one half hour
12.0 ml 200mM (100 X) L-glutamine
1.2 ml 50 mg/ml gentamicin reagent
Filter sterilize through a 0.22 祄 cellulose acetate filter and store up to 2 weeks at 4 degrees C.
Wash media: (1 liter)
To 1 liter of sterile RPMI 1640 with 2mM L-glutamine, add:
10.0 ml 2.5 M (100X) Hepes buffer
1.2 ml 50 mg/ml gentamicin reagent
Filter sterilize through a 0.22 祄 cellulose acetate filter and store up to 2 weeks at 4 degrees C.
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