Mircea, My lab sorts viable cells 3-4x per week. Although, we have different instruments (Elite-ESP cell sorter with an autoclone) our viability's and yields are excellent. This was not always the case and through much trials and tribulations we have several rules which can not be changed if you expect a successful sort. 1. pH your media, although not always an issue this will send cells over the edge. 2. Determine the rate of apoptosis in your culture before sorting. This is not complicated and does not need to be accurate (i.e. use of TUNEL or Annexin) you can simply plot the percentage of low angle cells vs. culture time. As it turns out some hard-core culture folks grow out their cells to far were they start to rapidly die. 3. As cells are sorted they will need to contact the same growth media which they started in. In addition to adding this medai into the tube the sides to the tubes or wells should be coated with this media prior to sorting. 4. Sort with a clean system. After removing your flow tip and replacing your sheath filter with a new filter we run 10% hypochlorite (d-HOH) for 3 mins. This is followed by distilled HOH for 3 mins. Finally, after replacing the tip, RPMI containing gentimycin is used for the sheath fluid thoughout the sort. Happy sorting. Never met a cell we could not sort! Stephen P. Perfetto, MS.,MT. (ASCP) Walter Reed Army Institute of Research Department of Molecular Diagnostics and Pathogenesis 1600 East Gude Drive Rockville, MD. 20850 _______________________________________________________________________________ Subject: how to get good viability after cell sorting? From: Mircea Podar <mpodar@ems.salk.edu> at Internet_Gateway Date: 10/19/99 5:38 PM Hello, I am having big problems with the survival of lymphocyte cells (CEMX174, B-T hybridoma) after single cell sorting. The cells are not stained (sort by scattering), so they they should be the happiest. However, less than 20% of the sorted cells survive. Here are the parameters that I tried, without success: -sorting using a FACS Star Plus (BD), tried both the 80 and the 100 micron nozzles, as low pressure as possible. The sheet fluid is the BioSure PBS (I noticed that many peple say that this is the best for high cell survival). -cells in either PBS+2%serum, buffered with HEPES or in RPMI+serum+HEPES (no phenol red). -cells deposited in medium, in medium with extra serum (up to 50%) or even in 100% serum. Also tried conditioned medium (medium filtered from the same cell type, in case they secrete a required factor). We know from serial dilution experiments that cells can survive if only one/well. Does anyone have any suggestions? Or maybe some cells simply cannot recover after sorting? Thank you! Best regards, Mircea ******************************* Mircea Podar, Ph.D. The Salk Institute, Infectious Disease Laboratory 10010 N. Torrey Pines Rd. La Jolla, CA 92037 Tel: (619) 453-4100 ext.1338 FAX: (619) 554-0341 e-mail: mpodar@ems. salk.edu *******************************
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