Hi Flow people I'm working on cord blood phenotyping, interested in rare leukocyte subsets. As those who work on these preps will know, there is a problem with RBC and erythroid contamination, variable but up to 80% of ficoll separated PBMC. They don't seem to lyse easily using hypotonic methods, and as some are nucleated, I wouldn't expect this to be an ideal approach. Gating on FS/SS can partly improve percentage of CD45+ cells but not much. We really want to clean the preps up, and I have been thinking of using complement lysis of the RBC, perhaps with an antibody to Glycophorin A. There are antibodies of different isotypes available, including IgM, I presume this might be the best to start with for complement lysis. I believe GlyA has a complement regulatory function, I don't know if this will be a problem. Does anybody have any hints on this, either the lysis method, or is there a simpler way of removing the erythroid cells that I have missed out on? Immunomagnetic separations can get a bit expensive, and positive selection of CD45+ cells will cause problems with further analysis. I presume there must be a lot of people doing this sort of thing, because of the CD34 interest. Thanks. ______________________________________________ Dr William Smith Institute for Child Health Research (Company Limited by Guarantee ACN 009 278 755) Subiaco, Western Australia, 6008 PO Box 855 West Perth WA 6872 Ph 61 8 9340 8792/8388, Fax 61 8 9388 3414 email williams@ichr.uwa.edu.au ______________________________________________
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