Figure 1

Very few early-minted ASC (plasmablasts) divide. (a) Representative flow cytometric analysis of Ki-67 expression in blood ASC at day 0, 1, and 3 in culture (with unstimulated naïve B cells at day 1 as a non-proliferative control). The numbers indicate the percentages of the cells positive for Ki-67. (b) Quantitation of Ki-67⁺ blood ASC (n = 2 independent biological replicates). 0, 1, 3, day in culture. (c) Representative flow cytometric analysis of BrdU incorporation in blood ASC at day 1 in culture after 3.5–4 h or 24 h pulsing. (d) Quantitation of BrdU⁺ blood ASC (n = 3 independent biological replicates). h, hours BrdU pulsed. All cells were assayed at day 1 in culture. (e) Quantitation of apoptosed blood ASC after 3.5–4 h or 24 h BrdU pulsing (n = 3 independent biological replicates). PBMC were used as control populations. 4, 24, hours BrdU pulsed. All cells were assayed at day 1 in culture. (f) Representative flow cytometric analysis of BrdU incorporation in blood ASC at day 1, 3, and 7 in culture after 3.5–4 h pulsing (with apoptotic cells excluded). (g) Quantitation of BrdU⁺ blood ASC (n = 4 independent biological replicates). All 3.5–4 h BrdU pulsed. In (c,f): Individual cell cycle phases (S, BrdU⁺; G0/G1, 2N and BrdU⁻; and G2/M, 4N and BrdU⁻) were indicated and the percentage of ASC in S phase was estimated by the amount of BrdU detected. Apoptotic cells were excluded from the analysis. (h) Image series displaying a single ASC with ongoing Ig secretion showing evidence of division (bright field) from day 0 to 21. (i) Zoomed in bright-field image series for direct visualization of the dividing cells from (h) on select days in culture. (j) Quantitation (left y axis) and frequency (right y axis) of dividing and non-dividing single cell blood ASC across 15 independent experiments. Div, dividing; Non-div, no dividing. In (h,i): Ref., reference image for visualization of the number of cells penned at loading.