Dealing with autofluorescence

Tony Schountz tschount at mesastate.edu
Thu Jul 26 10:13:38 EST 2001


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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