Figure 111.

Representative gating strategy and analysis of human bone marrow PC. (A) Analytical gating strategy. Time/CD45 visualization confirms the stability of the cytometric measurement over time. Time frames showing discontinuous data were excluded if applicable. As PC exhibit particular light scatter and background fluorescence properties, the CD138⁺CD38^high PC population was gated first, followed by cell aggregate exclusion and gating on CD3⁻, CD16⁻, CD10⁻, CD14⁻ and DAPI⁻ cells for exclusion of dead cells and potentially contaminating cell types. Then, the FSC-A/SSC-A plot reveals that PC show a broader light scatter signal distribution compared to other lymphocytes due to their increased size and ellipsoidal shape. Should the FSC-A/SSC-A plot reveal remaining FSC^low and/or SSC^low cell debris or electronic artifacts, these should be excluded by gating at this step. (B) Human BM PC consistently display distinct populations with either high or low to no expression of CD19 [901, 1105]. The absence of HLA-DR expression confirms at large the absence of PB [941, 1084], and remaining HLA-DR⁺ PB are excluded. (C) Backgating analyses of the procedure shown in (A). (D) Comparison of Ab staining and light scatter properties of total CD138⁺CD38⁺ BM PC vs total BM mononuclear cells. PC exhibit increased background fluorescence signals compared to other cells (possibly integrating cell size effects, autofluorescence, and non-specific binding of labeled Abs) stressing that gating should be adjusted at the level of PC rather than at global levels. Consistent with their increased size, non-spherical shape, and high organelle content, BM PC show a FSC / SSC pattern distinct from that of other BM cells.