As Dick says, BREfeldin doesn't fluoresce; besides, if it did, the signal would not be equal on all four detectors. If the background appears on all detectors it is more likely to be a matter of scatter than of actual fluorescence. This probably means either (a) you have some cell debris - removing it might be accomplishable by passing the cells through a Sepharose 2B column and taking only the front that passes through (i.e. don't be greedy), or (b) too high a flow rate so that you are getting multiple cells per drop, and/or (c) too high a concentration of cells - try using fewer per ml (we had to do this with platelets to get good patterns, run them at 10-30 x 10exp 5 per ml). I hope this helps. Richard Haugland wrote: > Pure brefeldin A should have no absorbance above about 260 nm and be > totally nonfluorescent. It has no real bond conjugation. > > [Structure: 2KB] > > > > Bill Justice wrote: > >> I have an investigator that is treating cells with BREfeldin A and >> then examining various intracellular and surface markers. We are >> getting significant "background" activity or more accurately >> distinct peaks in a "dim fluorescence" location amounting to around >> 8-12 %. This activity occurs in all four detectors in the same >> location and around the same amount. I am wondering if BREfeldin A >> fluoresces from a 488nm laser? Any thoughts? Thanks in advance. >> Bill Justice >> Laboratory Information Systems >> KU Medical Center >> Phone: 913.588.3876 >> Pager: 913.917.7018 > -- Elizabeth R. Simons, Ph.D. Professor of Biochemistry Boston University School of Medicine 80 E. Concord Street Boston, MA 02118 (617) 638-4332 (617) 638-5339 FAX esimons@bu.edu ersimons@earthlink.net
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