Hi all I have been trying to phenotype lymphocytes extracted from tiny gut biopsies, not surprisingly I am having some difficulties. I have used mechanical disruption methods (chopping up and sucking up and down needles) with success but as we want to do this with samples from HIV infected patients such an approach is inadvisable to say the least. So, I am now trying to use an enzymic method using collaginase and agitation for a couple of hours. The sample digests quite nicely but I am getting curious results like the majority of cells are CD19/CD3 positive! Some other markers are OK. I do my staining along side peripheral blood from the same individual which is fine. Incidentally I also tried using dispase to digest the tissue, worked fine but digests off CD4 and CD8. My question is does it make a difference which type of collaginase you use and can anyone think of another reason apart from low viability for this type of non specific staining, are there any better enzymes?. I am using four colours, Fitc, PE, PerCP and APC. Any suggestions? Protocols? Simon Monard Aaron Diamond Center New York
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