Hi Helen, You mention an intra-cellular stain, this usually requires treating the cells somehow to allow the stain through the membrane. Have you treated the isotype control samples the same way? Such treatments can change the baseline fluorescence... Cheers, Joseph. At 04:29 29/09/99, Helen Horton wrote: >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? -- Joseph Webster Flow Cytometry Facility Centenary Institute
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