Improving the characterization of endothelial progenitor cell subsets by an optimized FACS protocol
Fig 1
Basic strategies of sample preparation and FACS analysis.
A. FACS plots of Ficoll-enriched PBMCs vs. RBC lysis-based preparations. To prevent exclusion of large cells, a large mononuclear-cell gate was applied. B. Ratios of enrichment of total live PBMCs, lymphocytes, monocytes, HPCs (as defined in Fig 2) and CECs (as defined in Fig 3) by Ficoll vs. RBC lysis. C-D. Examples of reduction of background (auto-fluorescence) by stringent exclusion of dead cells using Hoechst 33258 (C) and (residual) granulocytes (D). E. Basic procedure used, in addition to standard acquisition (1x106 total events), to enrich for target cells by discarding triple CD34/CD133/KDR negative events. Upper left plot: selection of CD34+ and KDR+ cells; lower left plot: selection of CD34+ and CD133+ cells. Right panels: result of a “Join (Or) gate” in FACS DIVA. KDR+ CD34+ (upper plot) and CD133+ CD34+ (lower plot) populations are better visualized. F. Delineation of a scale of FACS-based fluorescence levels. Negative peak = unstained or isotype control; low (dim) = within 1 log from negative; high (bright) = more than 1 log from negative and ranging from moderately high (1st to 2nd log above negative) to very high (3rd log or higher). “Medium” is sometimes used to define populations in between low and high.