Supplementary Figure 2 from Tumor Endothelial Markers Define Novel Subsets of Cancer-Specific Circulating Endothelial Cells Associated with Antitumor Efficacy

PDF file - 86KB, Quantitative analysis of CTEC and CEC/CEP populations in human specimens by eight-color flow cytometry. A, Syto16 (DNA) gate used to exclude apoptotic, necrotic debris and cellular degraded fragments. Syto16-positive cells (red and blue dots) indicate all viable portions in peripheral blood mononuclear cells (PBMCs) specimen. B, P1 gate with SSC/FSC to display major blood components of the Syto16+ PBMCs (green dots). C, CD45-negative cells gated on Syto16/P1 to specifically select non-hematopoietic cells (blue dots) for further phenotypic analysis and quantitative determination. D. CD133 rectangle gate made on Syto16/P1/CD45-negative population to separate CD45-/CD133+ (pink dots) and CD45-/CD133- (blue dots) subsets. E and F, CECs/CEPs gated on Syto16/P1/CD45-/CD133+ or Syto16/P1/CD45-/CD133- subsets respectively, illustrate staining negatively or dim for hematopoietic marker CD45, positively for SYTO16 (DNA), endothelial markers CD146 and CD31, and negatively for the progenitor marker CD133 (pan dots). CEPs expressed the same phenotype as CECs except they were positively staining for CD133 (pink dots). G, Quadrant gates made on Syto16/P1/CD45- to identify the subsets expressing CD276+/CD133- (blue dots) for further phenotypic analysis and CTECs quantitative determination. H, CTECs gated on Syto16/P1/CD45-/CD133-/CD276+ demonstrate staining negatively or dim for hematopoietic marker CD45, positively for SYTO16 (DNA), tumor-derived endothelial marker CD276, endothelial markers CD146 and CD31, and negatively for the progenitor marker CD133 (aqua dots).