>
> 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