William,, C' can be a bit of a pain sometimes - activity must be titered and it's quite labile.It's also more expensive than NH4Cl lysis - but perhaps less membrane altering. I'm wondering more about the cytotoxicity of the GlyA. I would think one would need an IgG - those tend to be good C' fixers. If this could work it might provide a nice way to look at WBCs without subjecting them to the rigors of a shocking solution. It would also perhaps help rid CD34+ stem cell preps of scatter similar erythroblasts. >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 >______________________________________________ Barry Grimes Manager, Hematopoiesis FACS Laboratory UKMC Hematopoiesis Center 800 Rose St. Lexington,KY 40536-0093 606-323-8193 bagrim1@pop.uky.edu
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