From: D. Robert Sutherland (rob.sutherland@Utoronto.ca)
Date: Tue Dec 16 2003 - 16:42:02 EST
Hi Mattia, I've not done it, but the basic protocol should work OK. You could send me a couple of listmode files of an example of your methodology and I could take a look at them. Regarding the other question, are you interested in thy-1 and c-Kit expressing subsets of murine CD34+ cells, or did you mean 'can the same boolean gating strategy be employed to identify CD90 and c-Kit+ stem cells directly?'. Unless your antibodies to the latter antigens are relatively 'bright' and 'clean', it might be quite difficult, without first identifying bona fide CD34+ cells. We have atttempted to standardise the subsetting of CD34+ cells in human CD34+ cells (see Current Protocols in Cytometry Unit 6.4, or the old ISHAGE web site - it contains downloadable templates and listmode files for Coulter and BD instruments (www.celltherapy.org/committees/Committees/Graft_Evaluation/graft.htm) and I would think that this methodology would be applicable to the murine system. Good luck Rob mattiaqu@tin.it wrote: >Dear all, > I'm using the ISHAGE procedure (CD45/CD34 staining plus side >and forward scatter evaluation) to count CD34+ cells in mouse bone marrow. > >Are there any references that you know or can you tell me your experience >about using this procedure in mice? > >Do you think that a similar procedure would also be applicable for others >stem cell markers such as c-kit or thy-1? >Would it be reliable? > >Any advice will be appreciated > >Best Regards, > >MATTIA QUARTA M.D. >Department of Medical and Surgical Sciencies >University of Padua >Via Giustiniani 2 >35100, PADOVA >Italy >Phone: +39-049-8211873 >fax: +39-049-8211884 > > > >
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