Archived Protocol

<h3>Cell Culture Media and Solutions</h3> Dec. 18, 1990 R. Veile <hr><ul> Antimycotic/antibiotic media:<ul> 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.</ul> 1X Cyclosporin media: (100 ml)<ul> To 100 ml of growth media add: 1.0 ml 100X Cyclosporin A</ul> 2X Cyclosporin A media: (1 µg/ml)<ul> To 100 ml of growth media add 2 ml of 100X Cyclosporin A. </ul> 100X Cyclosporin A: (100ml)<ul> 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.</ul> Freezing media: (1 liter)<ul> 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) -------- 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. </ul> Growth media: (600 ml)<ul> 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.</ul> Growth media: (1 liter)<ul> 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. </ul> Wash media: (1 liter)<ul> 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.</ul> </ul> <hr><center> <a href=http://hdklab.wustl.edu/lab_manual/hcc/hcc10.html><img src=http://hdklab.wustl.edu/lab_manual/image/prev.gif></a> <a href=http://hdklab.wustl.edu/lab_manual/hcc/hcccontents.html><img src=http://hdklab.wustl.edu/lab_manual/image/main.gif></a>