Maciej, Intracellular cytokine staining is a lot trickier than surface staining. I have found that when I did IL8 on human PMNs my isotype control was brighter than my IL8 even though I knew by ELISA of cell lysates that the cells were positive! Irrelevant isotype means an antibody raised against something that is not found in or on human cells. It used to be KHL but it was latter found that in rare cases people had allergies to shellfish. A better control for intracellular staining of cytokines is the permeabilized cells preincubated with unlabeled antibody followed by labeled antibody. It is assumed that all the specific sites will be blocked by the first antibody so that the labeled antibody will be labeling the nonspecific sites. Also with B cells there are Fc receptors so it is wise to block these first with IgG before staining. Hope this helps. Margaret >Dear group, > >I've been recently having some problems with control antibodies for >the intracellular staining. Basically in my tubes with permeabilized >monocytes, the control glows in the 2nd log. > >The same cells, in a non-permeabelized tube, with another reagent >for isotype control, ie. surface staining control, is fine (no >signal). > >What I tried doing is using PE-conjuaged anti-CD19 antibody (not >found on mono's), which works great as a control - but I'm not sure >if this is a good reagent for intracellular staining? > >Sometimes I read people's materials and methods "irrelevant >isotype-matched fluorescent antibody was used.." what exactly does >that mean? > >thanks for any suggestions, > >maciej > >===== >`---------------------------------------------` >| Maciej S. Simm | 525 E 68th Street | >| Research Technician | Room N-805 | >| Cornell Medical Center | Tel. 212.746.3428 | >`---------------------------------------------` >|www.cd4cd8.com | 917-734-6280 | AIM - XsimmX | >`---------------------------------------------` > >__________________________________________________ >Do You Yahoo!? >Get email at your own domain with Yahoo! Mail. >http://personal.mail.yahoo.com/ >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> Margaret M. Tropea Critical Care Medicine Department National Institutes of Health (ph) 301-496-7752 (fax) 301-480-3389 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>
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