Clone mi15

Cultured cells (5× 10⁴ - 5× 10⁵) were cytocentrifuged onto poly-L-lysine-coated slides (for 5 minutes at ~120 g; Cyto-Tek centrifuge model 4332; Sakura Finetek, Japan). Slides were air-dried for 1 hour at 22°C and then fixed in 4% paraformaldehyde at 4°C for 10 minutes. Subsequently, they were blocked with 2% bovine serum albumin in PBS and incubated with Hoechst 33342 solution (2 μg/mL, Cat. B2261; Sigma-Aldrich) to stain DNA. We stained for plasma cells with mouse anti-human CD138 mAb (1:250, Clone MI15, Dako), donkey anti-mouse IgG Alexa 594 (1:300, A21203; Molecular Probes-Invitrogen) and goat anti-human IgG Alexa 488 (1:500, A11013; Molecular Probes-Invitrogen). Slides were mounted in 80% glycerol-TBS and stored at 4°C in the dark. All washing and incubation steps were performed with TBSTriton X-100 (0.03%). We counted the plasma cells from each well, in a blinded fashion, on a fluorescence microscope (Olympus BX51), identifying them by their distinctive size, shape (extensive cytoplasm and eccentric nuclei), and positive staining for internal IgG and/or surface CD138. Absolute numbers of plasma cells per well are given in Table 2 and shown in the Figures as the % of those in untreated cultures. Total numbers of recovered cells were measured by automated counting of trypan blue-excluding cells (TC20 Automated Cell Counter, BioRad).