Re: Cord blood
Pete Macardle (Peter.Macardle@flinders.edu.au)
Fri, 1 Aug 1997 08:40:50 +0930 (CST)
>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
>>______________________________________________
>>
>>
>>
>>
>