Hey all, I have a client who is staining for intracellular cytokines (IFNg and IL-4) and surface staining for CD4 and CD8 in human BAL. Before activating cells with PMA and ionomycin (plus Golgi Stop), the cell surface markers look fine. However, when we activate the cells and stain for intracellular cytokines, CD4 is down regulated to a point where it is virtually indistinguishable from the negative population. CD8, IFNg and IL-4 look great, but CD4 goes away. We've tried two different clones and 3 different fluorochromes (APC, FITC and Alexa 488) for CD4, but is always the same result. We've thought about using Flow Amp, but haven't ordered it yet. Also, we don't see this phenomenon when performing the assay on PBMC. It seems to be unique to BAL. Has any one out there in Flow Land experienced this scenario and have any ideas on what may be happening and how to resolve it? Thanks all, Rick Peppler Flow Cytometry Shared Facility Medical University of South Carolina Charleston, SC
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