Dear Jim, The reagent cocktail of choice for the purpose of counting Thy-1+ CD34+ subsets in PB, apheresis or marrow samples is CD45PE:Cy5, CD34FITC and Thy-1PE. We have purified the CD45 antibody secreted by the 4B2 clone (from the ATCC) and had it custom labelled with PE:Cy5 by Southern Biotechnology (1 800 722 2255) and it works well. Alternatively, the J33PE:Cy5 clone from Coulter/Immunotech is very good. The CD34 antibodies have to identify 'class III' CD34 epitopes since class II antibodies do not work after FITC conjugation (see The ISHAGE Guidelines For CD34+ Cell Determination By Flow Cytometry. J. Hematotherapy 3: 213-226, 1996). 581FITC (Immunotech/Coulter) or HPCA2FITC (BD) work very well. Since the Thy-1 antigen is not expressed at high levels on candidate hematopoietic stem cells (CD34bright), FITC conjugates are not recommended. We have had extensive experience in this assay with PE-conjugated 5e10 (from Pharmingen) (see Optimizing the CD34+ Thy-1+ stem cell content of peripheral blood collections. Exp. Hematol. 23:1619-1627, 1995). While Pharmingen have recently introduced a PE:Cy5 conjugate of this clone, we have not yet fully evaluated it but it looking quite promising so far. If it turns out to be as good as the PE conjugate, one would also be able to use a CD45FITC/CD34PE/Thy-1PE:Cy5 cocktail with confidence. This cocktail might indeed be the best since one is utilising the two brightest fluorochromes (PE and PE:Cy5) to detect the two weakest antigen systems (CD34 and Thy-1/CD90). Hope this is helpful, Rob Sutherland, ISHAGE Stem Cell Enumeration Committee, Oncology Research, The Toronto Hopital.
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