On Thu, 31 Jul 1997, Dr William Smith wrote: > > 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 > ______________________________________________ > > Try using 42 degree NHCL lysing buffer for 5-10 min. Spin down in a 4 degree centrifuge for 5 min. Good luck. Jim
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