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CD34+ hematopoietic stem progenitor cells detected in peripheral blood of CHB patients by flow cytometry using ISHAGE logical gating strategy. Small aliquots of PBMCs were incubated with PE-conjugated anti-CD34 antibody, vioblue-conjugated anti-CD45 and analyzed by flow cytometry. (A) representative plots of whole MNCs shown by CD45 vs. side scatter. The initial gate P1 is set to include all CD45+ events including CD45dim and CD45bright; (B) the events in gate P1 are then displayed on a CD34 vs. SSC dot plot and a second gate (P2) produced to include the cluster of CD34+ events; (C) this plot is obtained by plotting the events that fulfill the criteria of gates P1 and P2 (sequential gating), Cells forming a cluster with characteristic low SSC and low to intermediate CD45 fluorescence are then gated on this third plot to produce a region (P3); (D) events fulfilling the criteria of all three gates (P1, P2 and P3) are then displayed on a FSC vs. SSC dot plot to confirm that selected events fall into a ‘lymph-blast’ region (P4); (E) ungated data are displayed on a CD34 vs. CD45 histogram to establish the lower limit of CD45 expression by the CD34+ events; (F) region (P5) is set to include events no smaller than lymphocytes by back scattering a small number of lymphocytes from plot 1 (high CD45 staining with low side-scatter). Any events falling outside region P5 are excluded from final calculation. PBMCs: peripheral blood mononuclear cells; T: total events acquired.
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