How about first staining cells with (for example), FITC-anti CD22. Then do the fix and perm, staining with (for example) PE-anti CD22 (of the EXACT same clone!). Then you get all the information you want: membrane CD22 (FITC signal), cytoplasmic CD22 (PE signal), and of course, their ratio. You must use the same clone for competition reasons. You'll have to verify that you don't lose FITC signal during the fix & perm + PE staining step--easy enough controls--I would worry some about the PE antibody replacing the FITC antibody. But it's likely to be straightforward to set this up. If you don't want to use up a color, then just pre-stain with unconjugated CD22 to block off the surface antigen. mr At 8:32 AM -0500 9/10/01, qli@sbmflab.org wrote: >I need some help. I am trying to determine the cytoplamic CD22 in >B-Lymphocytes using Caltag's FIX &PERM reagent. Since CD22 also expresses >on the surface of B-Lymphocytes, how to find a method which only measures >cytoplasmic not surface CD22? Also, how can I confirm that I am measuring >cytoplasmic CD22? Thanks.
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