Hi, I a work with surface staining of CD4 and CD8, followed by intracellular staining of INFg in T cell stimulated with anti-CD3/28 from PBMC. This is the general protocol I used: 1) Wash the cell with PBS, 2) Block with 2% BSA, 3) surface staining with CD4 and CD8 for 30mins; 4) Wash the cell with PBS twice, 5) Permeabilize the cell with 1x permeabilization buffer by centrifugation twice(5mins each), 6) intracellular staining with INfg for 30mins in 1xpermeabilization (RT, dark, 1% BSA), 7) Wash with 1xpermeabilization buffer, 8) FACS analysis.
I tested this protocol in PBMC cell, with very successfull staining of CD4,CD8 and INFg, but when using T cell, only INFg could be stained. I already failed several times in this assay. So why the surface staining of CD4 and CD8 were both failed? It seems there is some problems in this protocol. Would it be the order of Fixation and Surface staing? Like that, some staining antibodies (CD4?) may favor fixation prior to surface staining?
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