>Date: Fri, 01 Aug 1997 08:37:07 >To: Dr William Smith <williams@ichr.uwa.edu.au> >From: Pete Macardle <Peter.Macardle> >Subject: Re: Cord blood > >HI William > >We published in J Imm Methods 195 43-48 1996 concerning this problem. You can usually get good preps after a dual ficoll/hypaque separation. We have just submitted a paper with an improvement usin a glycophorin antibody that agglutinates the RBC, including the nucleated, and allows for very clean preps. > >Anyone who wants full details please contact direct. > >Best wishes >Pete > >At 01:10 PM 7/31/97 +0800, you 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 >>______________________________________________ >> >> >> >> >
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