I've seen something similar happen with two sets of single-labeled cells (one FITC and one PE on an antibody against the same antigen) that were mixed together and examined over time. In 10min there is a half log increase in FITC fluorescence. If the size of the newly bright cells is examined, their forward light scatter is larger suggesting that cells were sticking together. Whole antibodies were used suggesting that Fc binding could be a problem. A test such as adding protein A or using F(ab)' fragments was not done to test the hypothesis. Only PE-labeled cells were found to pickup FITC label suggesting that the larger PE protein prevents cell-cell cohesion. Dave ======== David M. Coder, Ph.D. Director, Cell Analysis Facility Univ. of Washington School of Medicine 1959 NE Pacific Street H474A Box 357650 Seattle WA 98195 tel. 206-685-3014 email: dcoder@u.washington.edu ----- Original Message ----- From: Maciej Simm <simmmmer@yahoo.com> To: cyto-inbox Sent: Tuesday, March 13, 2001 10:17 AM Subject: with time, geometric mean increases? > > Dear group, > > I had an unusually large batch of monocyte cytokine stains to do > yesterday, and the acquisition took a lot longer than usually. Long > enough, in fact, to notice that between the control sample (acquired > at time=0) and the last patient (acquired at time=3.5 hrs) CD33 FITC > and CD45PE-Cy5.5 signal went up by half a log. > > The samples were fixed and washed multiple times so there's no way > there was any free antibody left to "add" more signal. > > Why did the signal go up so much? > > The unstained, external and internal isotype stains were unaffected. > > Any thoughts would be appreciated, > > Maciej > > __________________________________________________ > Do You Yahoo!? > Yahoo! Auctions - Buy the things you want at great prices. > http://auctions.yahoo.com/ >
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