Re: Thy1/CD34 staining
adurett@notes.mdacc.tmc.edu
Mon, 31 Mar 1997 11:04:08 -0500
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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