Cell Culture Media and Solutions

R. Veile

Antimycotic/antibiotic media:
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 µm 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 µg/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 µl ) 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 µm 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 µm 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 µm 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 µm 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 µm cellulose acetate filter and store up to 2 weeks at 4 degrees C.