Figure 2.

Flow cytometer strategy for CPCs. CPCs are defined as CD45^dim/CD34⁺/CD133⁺ using CD45-FITC, CD34-PE Cy5, CD133-PE conjugated antibodies. Gating strategy for peripheral blood CPC, a modification of the International Society for Hematotherapy Graft Engineering stem cell strategy. (A) All CD45⁺ events are displayed on a side scatter (SSC) versus CD34 plot. G1 events include the CD34⁺ cluster with low SSC. (B) G1 events are displayed by CD45 versus CD133 scatter plot. Cells demonstrating positive CD133 fluorescence and dim-to-intermediate CD45 expression (G2) are gated onto a third plot. (C) G3 displays events from G1 + G2, with characteristic forward scatter (FSC) and SSC. The lower limit of FSC of G3 is determined by backscattering lymphocytes (R1 + L1), so that G3 only includes events no smaller than lymphocytes. CPCs are defined as CD45^dim/CD34⁺/CD133⁺ cells with characteristic FSC/SSC. (D) Ungated population demonstrates FSC/SSC of mononuclear cells of low SSC (majority probably lymphocytes) R1 population. (E) CD34 versus CD45 plot of R1 demonstrating region L1 (CD34-ve leukocytes), the clear CD34⁺ events (R2), and the establishment of the lower limit of CD45 expression by CD34⁺ events. (F) SSC versus CD34 plot of CD45⁺/IgG₁-PE Cy 5 events (isotype control).