Plasma cell apoptosis is linked to excessive ER stress. (A) Side scatter analysis of organelle density (top panel) and staining of the secretory apparatus with BFA-BODILY (bottom panel) in in vitro–differentiated plasma cells and I.29μ⁺ cells. Plasma cells were analyzed immediately after purification (d0) and after 2 days in culture. I.29μ⁺ cells were analyzed before (d0) and 1 to 4 days after LPS stimulation. annexinV-FITC negative cells only were analyzed in the side scatter analysis to exclude nonspecific effects on light scattering in dying cells. Data are representative of 3 independent analyses (2 in in vitro–differentiated plasma cells). (B) Analysis of ER chaperones BiP and GRP94, IgM, and GAPDH (control). Immunoblotting was carried out on whole cell extracts prepared from in vitro–differentiated plasma cells at the time of purification (d0) and 2 days later, and I.29μ⁺ cells before (d0) and 1 to 4 days after LPS stimulation. (C-D) The normalized proportion of dead cells/untreated control (see Figure 2C) after treatment with the indicated inducers of apoptosis was determined by staining with annexin V–FITC and propidium iodide (mean and SEM of 2 independent experiments). (C) Primary ex vivo–isolated resting B and plasma cells were analyzed after treatment with 1 μg/mL tunicamycin for 14 hours. Viability of untreated controls is identical to Figure 2C. P values refer to the comparison of treated with control cells, and asterisks indicate statistical significance. (D) I.29μ⁺ cells 1 to 4 days after LPS stimulation were treated with tunicamycin as in panel D, DTT (1mM for 4 hours), etoposide (1 μg/mL for 16 hours), or arsenic trioxide As₂O₃ (1μM for 24 hours). Death rates of untreated controls are equivalent to those shown in Figure 1A and P values refer to the comparison between relative death rates on day 1 and day 4.