Anti bcma

For coculture of isolated splenic B cells with TANs, 0.5 Â 10 6 of splenic B220 þ B cells (isolated from spleens of tumor-bearing mice) were mixed with 0.5 Â 10 6 of isolated TANs per well in 96-well plates, in 250 mL complete RPMI media, in the absence or presence of anti-BAFF-R (15 mg/mL), anti-TACI (15 mg/mL), or anti-BCMA (25 mg/mL; all R&D Biosystems) at 37 C for 1 hour, prior to adding neutrophils. Following overnight coculture, cells were harvested and centrifuged at 1,280 rpm (4 C for 6 minutes). Supernatants were collected for further quantification of IgG concentration using a Mouse IgG ELISA Kit (ab157719, Abcam) according to the manufacturer's instructions. One hundred microliter of supernatant either nondiluted or diluted 1:5 in diluent buffer (provided by manufacturer) was used for each well, and duplicates were made for each sample. Concentrations were calculated based on a standard curve within the range of 0 to 200 ng/mL and adjusted in case of dilution. Color development was monitored at 450 nm within 10 minutes following addition of the stop solution, using Tecan Spark microplate reader.
Pelleted cells were resuspended in FACS buffer (1X PBS, 2% FBS, 2 mmol/L EDTA, and 0.1% NaN 3 ), stained with APC-conjugated anti-mouse B220 (Biogems) and PE-conjugated anti-mouse CD138 (Miltenyi Biotec), and analyzed via flow cytometry (described above).