Greetings all! I am relatively new to the flow cytometry field and am experiencing some problems with compensation. I am surface staining with CD4-PerCP, CD69-PE and intracellular staining with IFN-gamma-FITC. The staining is performed on lysed fresh blood samples. I set the PMTs on isotype controls with no problem but when I try to perform compensation the PE and Per-CP antibodies appear to be staining negatively- all squished up on the axes, and no amount of messing with the FL channels will get them off the axes. I thought that something may be quenching the fluorescence and tried additional washing steps after lysing the RBCs but am still having the same problem- has anyone had this problem before or does anyone have any suggestions? Thanks in advance, Helen Helen Horton PhD Wisconsin Regional Primate Research Center 1220 Capitol Court Madison WI 53715 Tel: (608) 265 3381 Fax: (608) 263 4031 hhorton@primate.wisc.edu
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