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Method: Removal of Yeast Contamination from Lymphoblast Cultures

May 30, 1990

Rosalie Veile


Purpose:

Procedure:

  1. Pipet 5 ml histopaque into a 15 ml centrifuge tube.
  2. Carefully layer on top of histopaque 10 ml of contaminated culture (or concentrated cells/yeast resuspended in growth media).
  3. Centrifuge tube for 25 minutes at 2500 rpm (no brake) at room temperature, using the TJ-6 centrifuge.
  4. 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.
  5. 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.
  6. 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:

  • 1X Cyclosporin media: (100ml)
  • 100X Cyclosporin A: (100ml)
  • Antimycotic/antibiotic media:
  • References:

    Biotechniques, Overhauser, Joan, et al. Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA.