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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