Tony, Dead and dying cells have higher autofluorescence - try resuspending your cells in 1 ug/ml propidium iodide (diluted in your staining media; I recommend RPMI without phenol red + 3% serum for better viability [other things in RPMI can contribute to autofl when staining with mABs - write me if interested in the formulation we and many others use]) and gating out PI+ cells in FL3. Also, larger cells (macs and grans) have higher autofluorescence than lymphocytes - so even if you have great viability you may still have the diagonal "tail". Try using scatter gating to focus on your cells of interest. Good luck! Rachel ======================================================= Rachel M. Gerstein, Ph.D. Department of Molecular Genetics and Microbiology Graduate Program in Immunology/Virology University of Massachusetts Medical School 55 Lake Avenue North Worcester, MA 01655-0002 (508) 856-1044 (508) 856-5920 (FAX) > ---------- > From: Tony Schountz > Sent: Thursday, July 26, 2001 11:13 AM > To: Cytometry Mailing List > Subject: Dealing with autofluorescence > > I'm having a bit of a problem that I'm not sure how to deal with. If I run > freshly isolated unstained mouse spleen cells through our FACScan, the > cells autofluoresce equally into FL1 and FL2, to about 10^1.5 or so. In > other words, there seems to be a perfect diagonal of a few cells that > autoflouresce (the majority are below 10^1). Gating on any population does > not affect this pattern. I prep the insturment using Calibrite beads, but > have not tried adjusting any of the settings to fix this. I've harvested > spleens in serum-free DMEM (with phenol red), serum-free HBSS (with phenol > red) or PBS and have washed 2x in 0.5%BSA-PBS, all with similar results. > Are there any suggestions as to how I can get this under control? > > Thanks, > > Tony > -- > Tony Schountz, Ph.D. > Dept. of Biology > Mesa State College >
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