At 13:10 97 - 07 - 31 +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
>______________________________________________
>
>
To remove the erythroid cells you may try the lysis method with
0.83 per cent ammonium chloride (0 - 4 oC) followed by washing
in PBS, provided the lysis reagent is not harmful to your cells.
Good luck
wp
++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++
Wladyslaw Pajdak
Dept. Clinical Immunochemistry, Skawinska 8, 31-066 Cracow, Poland
Int. Med. II, Collegium Medicum, Tel.: +48(12)56 54 25 ; +48(12)56 51 22
Jagiellonian University Fax: +48(12)56 57 86
E-mail:
mmpajdak@cyf-kr.edu.pl
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