Dear Colleagues, Happy New Year! I am posting a request for response for a user in my facility. Specifically, she is trying to quantitatively assess the apoptotic response of drug-treated patient PBMC lymphocytes (only) using an Annexin/PI kit. She cultures the PBMCs for a specified amount of time with IL-2 to activate them. She then ficolls to remove the dead cells before Annexin staining. When she brings them to me for flow analysis, we are seeing is a large population of lymphs, a small population of monos, and a population of what I believe to be non-lysed RBC's overlapping the apoptotic (various stages) cells from the PBMCs. This, of course, makes it difficult to distinguish the apoptotic lymphs from the RBCs and apoptotic monos using light scatter. I've suggested to this researcher that she find a common lymphocyte marker (preferably read in the FL3 detector) so that we can more specifically gate on only the lymphs. She asked me to post a message on the Cytometry list to see if there is anyone out there who is doing a similar assay who could give her some suggestions. So here it is. Any and all replies will be greatly appreciated. Also, I would be grateful for any suggestions on optimal acquisition techniques. Thanks for your time, Jodi Jodi L. Kroeger Coordinator, Flow Cytometry Core Facility Moffitt Cancer Center & Research Institute 12902 Magnolia Drive Tampa, Florida 33612 Office: (813) 979-6705 Lab: (813) 972-8400 ext. 2005 Fax: (813) 979-6700 kroegerjl@moffitt.usf.edu
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