Hi Kevin, The method I use to fix cells for later analysis is to use 2% paraformaldehyde. Basically you wash your cells after your last stain with PBS and then resuspend in about 200 uL of PFA made up in PBS. Some people wash this off after 20 mins to ½ h of fixing, but I’ve left my cells in the fix until I’m ready to run them (a bit lazy). If you are not using PI or any other viability stain, then how can you tell your cells are dead? Any fixative will cause some shrinkage of the cells which will make them appear smaller on your FSC v SSC profile. PFA is preferred over formalin because it preserves fluorescence whilst minimising background/autofluorescence. Hope this helps Joanna Joanna Kemp, PhD Animal Biotechnology Research Laboratories Monash University Australia _____ From: kevin xu [mailto:seaqy@hotmail.com] Sent: Wednesday, 30 April 2008 12:41 AM To: cyto-inbox Subject: Methods for fixing cells Hi, everyone: I'd like to fix my cells to run the flow later. The cells are human PBMC cells and are stained for surface markers only. PI will not be added and I will use FCS/SSC to gate out the living cells later. What kind of method I'd use? Can I use formalin instead of Para formaldehyde? I tried 5% formalin but there lots dead cells after fixing. Thank you very much! Qingyong Xu Department of Pathology and Laboratory Medicine Saskatoon Health Region and University of Saskatchewan Canada. _____ Express yourself wherever you are. Mobilize! <http://www.gowindowslive.com/Mobile/Landing/Messenger/Default.aspx?Locale=e n-US?ocid=TAG_APRIL>Received on Wed Apr 30 15:18:00 2008
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