Purpose:
This method is advantageous for saving the occasional cultures that become contaminated. Yeast contaminated cultures will appear cloudy when slightly shaken and lymphocytes will not cluster together as much as normal. If cultures are suspect, a drop of culture can be streaked on a YPD media plate to check for growth of yeast colonies, or a 5 ml sample can be taken to Barnes Diagnostic Center for identification of yeast strain.
Procedure:
Pipet 5 ml histopaque into a 15 ml centrifuge tube.
Carefully layer on top of histopaque 10 ml of contaminated culture (or concentrated cells/yeast resuspended in growth media).
Centrifuge tube for 25 minutes at 2500 rpm (no brake) at room temperature, using the TJ-6 centrifuge.
The yeast cells will pellet to the bottom of the histopaque gradient and the lymphoblast cells will be located on top of histopaque gradient. Remove the lymphoblast cells with a 10 ml disposable pipet, and transfer to a 15 ml centrifuge tube.
Wash cells by adding 10 ml of wash media to cells. Centrifuge 10 minutes at 1200 RPM, no brake, at room temperature. Aspirate off the wash media and resuspend in RPMI-growth media containing 1X antimycotic/antibiotic. This will remove the rest of the yeast cells.
Transfer the cells to a 25cm2 Tissue Culture Flask and feed the culture twice a week with 1X antimycotic/antibiotic media until all traces of contamination are gone. This will depend on the severity of the contamination (usually for cultures moderately contaminated, 2 weeks or 4 feedings will suffice). After contamination is no longer visible, feed the cultures with growth media containing only antibiotic and not the antimycotic.
Solutions:
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.
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.
1X Cyclosporin media: (100ml)
To 100 ml of growth media add: 1.0 ml 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 (or 20祃) of Tween 80 and mix well. While continually stirring, add 1 ml RPMI drop by drop. Quanitate 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.
Antimycotic/antibiotic media:
References:
Biotechniques, Overhauser, Joan, et al. Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA.
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.
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