Figure 1.

Flow cytometry strategy for CECs. CECs are defined as CD45^-/CD34⁺/CD146⁺ using CD45-FITC, CD34-PE Cy5, CD146-PE conjugated antibodies. Sequential gating strategy for CECs: (A) Forward (FSC) and side scatter (SSC) plot of white blood cells and gating region G1 to include all mononuclear and polymorphonuclear cell events while excluding platelets, dead cells, and microparticles. (B) Dump channel to exclude CD45⁺ cells and very high side scatter events. (C) Cellular events from gated region G2, with CECs in R1 (CD34⁺/146⁺). R1 cells are backscattered to ensure that they meet the minimal FSC requirements, that is, size equal to or greater than that of lymphocytes (Figure 2: CPC strategy CD where R2 indicates progenitor cells (CD34⁺)). CECs have been highlighted in bold for clarity. (D and E) Negative controls using fluorochrome-matched isotype control antibodies. (F) R3 illustrates small population of CD146⁺ leukocytes that are likely to be lymphocytes.