Our original evaluation of peripheral blood, PBPC, and marrow was performed using CD34(8G12)FITC from BD and CDw90PE from Pharmingen and reported in Blood86(7):2842-2848, 1995(Oct 1), Bone Marrow Transplantation 18:1073-1079, 1996, and Transfusion to be published summer of 1997. Subsequently, Pharmingen has commercially released their CDw90 clone conjugated with CyChrome(PE-Cy5) which we have evaluated and are currently using with CD34(8G12)PE from BD for progenitor cell evaluation. This combination has enhanced the separation of both the the CD34 dim and bright(Lin-) populations as well as the Thy dim, medium and bright subsets. We have also evaluated CD34(8G12)PerCP from BD in combination with Pharmingen CDw90PE and found it to be similar to the CD34(8G12)FITC combination. To ensure that we are collecting a statistically significant number of events and enhance the visualization of the subset separation, we routinely perform a live gate acquition of 65,000 events in a low SSC light scatter gate. These antibodies were titrated for a cell concentration of 10^^5 to 10^^6, with a saturation concentration occuring at 500ng; we routinely stain 5x10^^5 cells per three color panel, using logical gating to quantitate the total CD34 and the subsets of interest. Hope this will be of some help! April Technology Transfer - Flow Cytometry Blood & Marrow Transplantation Univ TX MD Anderson Cancer Center
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