Streptavidin conjugated microbeads

Three weeks after NP exposure, mice were sacrificed and the bone marrows harvested. The cells were labeled with a biotinylated anti-CD138 Ab (clone 281–2), followed by incubation with streptavidin-conjugated microbeads (BD Biosciences), and the CD138⁺ fractions were positively selected by magnetic separation (BD IMagnet). Both CD138⁺-enriched and -depleted fractions were collected, resuspended in PBS, and 2 × 10⁷ cells were transferred to mice via i.p. injections. Enrichment of cells was confirmed by FACS analysis of the enriched, depleted, and unfractionated bone marrow cells. CD138-allophycocyanin (clone 281–2) Ab and streptavidinallophycocyanin were used for staining and analysis at the UMMS and Tufts FACS core facilities. The data were analyzed using FlowJo software, and dead cells were excluded based on forward and side scatter. For bone marrow transplantation, C57BL/6 mice were treated once with 5-fluorouracil at 150 mg/kg body weight delivered i.p. 2 d before transfer. J_H^–/– mice receiving bone marrow cells were subjected to a total, whole-body dose of 5.0 Gy (500 rad, gamma ray source). Mice were challenged i.t. 2 wk following transfer.

B cells were purified (90–95%) by negative selection with single-cell splenocyte suspensions by using biotinylated anti-Thy1.2 mAb followed by streptavidin-conjugated microbeads (BD Pharmingen). In brief, 0.75 × 10⁶ cells/ml were cultured in 3 ml of RPMI 1640 medium (Life Technologies, no. 23400–021) in a six-well cell culture plate (Peak Serum, no. TR5000), supplemented with recombinant mouse BAFF (10 ng/ml). B cells were activated and plasma cell differentiation was stimulated using anti-CD40 (BD Biosciences, no. 553788) (1 μg/ml) or LPS (Sigma-Aldrich, L2630) (1 μg/ml), supplemented with IL-4 (PeproTech, no. 214–14) (10 ng/ml) and IL-5 (PeproTech, no. 215–15) (10 ng/ml) as indicated. RBN012579 was dissolved in 100% DMSO for stock solutions (1 mM; 368 μg/ml). Based on experimental data later reported in Schenkel et al. (33 (link)) (e.g., the section “Biochemical and pharmacokinetic characterization of RBN012759,” the Methods section, and legends to figures 4 and 5), cultures were treated with either DMSO or PARP14i (1 or 0.33 μM RBN012759), added once daily. Cultures were harvested and counted on day 5; supernatants were collected for ELISA to quantify relative levels of Ab (IgG1, IgG2a, and IgE) as described (14 (link)).