Detection of morphologic differences within B-cell compartments across different tissues using viSNE analysis. (A) viSNE analysis was performed on total live, single B lineage cells by using default parameters in Amir et al.⁴⁶  θ = 0.5, perplexity = 30; iterations = 1000. B cells from each donor were downsampled to 2000 events to assure equal weighting in the subsequent analysis. Concatenated fetal calf serum files from blood (n = 14 donors) and BM (n = 14) tissues were further downsampled to 22 000 cells to be equivalent to SP (n = 11). All markers from “Stain 1” (supplemental Table 2) except for the viability dye were included in viSNE clustering. The merged file is depicted at far left, followed by the individual tissues. Manual gate overlays are shown at the far right. Population definitions for manual gating are: naive B cell, CD19⁺ CD27^– CD95^– CD38^–; memory B cell, CD19⁺ CD27⁺ CD38^–; antibody- secreting cells (ASC), CD38^hi CD138⁺ CD27⁺; Ag-experienced, CD19⁺ CD27^– CD95⁺ CD38^–. (B) Heatmap profile of the individual markers as expressed in the concatenation of all 39 samples.