FLOW CYTOMETRY IN PRECLINICAL TOXICOLOGY/SAFETY ASSESSMENT - Flow Cytometry in Drug Discovery and Development - page 132

investigators choose to use a pan-leukocyte marker (i.e., CD45) rather than a panel for
leukocyte subsets, this is not recommended since there is disagreement in the
literature regarding CD45 status on progenitors (hematopoietic and endothelial).
Our department was also privy to unpublished observations from another laboratory
that observed CD45 þ immunohistologic staining of endothelium of select vascular
beds. Therefore, the minimum immunophenotypic definition used to identify putative
CEC/CEP became CD36 þ /HO þ /CD11b/c /CD3 /CD45R /CD42d /ASBMS .
CD3, CD11b/c, CD45R, and CD42d were combined into a single-color hematopoie-
tic lineage “dump” channel to allow room for other endothelial “specific” antibodies.
CD36 þ /Lin will be used for brevity throughout the rest of this chapter to refer to
the minimum immunophenotype. Representative flow cytometry data and sorting
strategy is described in Figures 6.2 and 6.3.
FIGURE 6.2 CEC flow cytometry gating strategy. (a) Debris is excluded from analysis based
on low forward and side laser scatter. “Intact cells” are gated to (b). (b) Cell aggregates are
excluded fromanalysis based on forward scatter pulsewidth. “Singlets” are gated to (c). (c)Only
HO þ cells are included in analysis. “Nucleated” cells are gated to (d). (d) CEC gate is tested on
CD36 cells recovered frommagnetic separation. This gate should bevirtually devoid of events.