Lora, Good luck, Tony Bakke Antony Bakke, PhD Director, Immunology & Flow Cytometry Oregon Health Sciences University bakkea@ohsu.edu >>> "Barsky, Lora" <LBarsky@chla.usc.edu> 08/25 8:53 AM >>> Can anyone offer insight as to why my users have been flow problems when using 293A and 293T cell lines on BD FACScan and BD FACSCalibur? The cell lines are adherent, so they trypsonize to release from the plates, they bring the cells in DPBS to FACS. They're all looking for GFP expression. I've suggested theyDPBS contains calcium and magnesium, which usually accelerate clumping. You might try adding EDTA or use Ca and Mg free PBS. Also keeping the cells cold will reduce clumping. Finally, many clumps are due to dying cells releasing DNA that traps other live cells. Adding a small amount of DNAase can solve this problem. pre-filter with a nylon mesh - 55um. But this doesn't help much. No other problems with flow on other sample types. Any info would be appreciated! Thanks, Lora W. Barsky CHLA Research Immunology/BMT
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