Anti cd19 pcp cy5

A total of 10⁶ PBMCs were plated in 96-well round-bottom plates in RPMI + 10% FBS (Gemini) + 1% penicillin, streptomycin, and l-glutamine (Invitrogen, Carlsbad, CA). After centrifugation and removal of media, cells were surface stained with 50 μL of a cocktail mix consisting of titrated volumes of LIVE/DEAD violet dye (Life Technologies, Grand Island, NY); anti-CD3, anti-CD14, and CD16 Pacific Blue; anti-CD19 PcP-Cy5.5; anti-CD27 APC; anti-IgD PE; anti-CD20 FITC; anti-CD24 PE-Cy7 (BD Biosciences, San Jose, CA); and anti-CD38 APC-AF700 (Beckman Coulter, Brea, CA) (table 2). A “dump” channel consisting of LIVE/DEAD dye and CD3, CD14, and CD16 Pacific Blue was used to exclude dead cells, T cells, and monocytes. After fixing the cells with 1% paraformaldehyde (PFA), they were acquired on a LSRII flow cytometer (BD Biosciences, San Jose, CA).

Following previously published methods,^{22 (link)} 2 × 10⁶ PBMCs resuspended in RPMI + 10% FBS + 1% penicillin, streptomcyin, and l-glutamine were stimulated with 10 μg/mL LPS (Sigma-Aldrich, St. Louis, MO) and 1 μg/mL CD40L (R&D Systems, Minneapolis, MN) for 48 hours at 37°C.^{22 (link)} Forty-eight hours allows for the detection of both B10 and B10 progenitor cells, which are cells capable of maturing into IL-10 competent cells after stimulation.^{22 (link)} For the final 5 hours of incubation, PMA (1 μg/mL, Sigma-Aldrich), ionomycin (0.25 μg/mL, Sigma-Aldrich), and brefeldin A (1:1,000 dilution, BD Biosciences) were added to the wells. After this period, cells were collected and stained with 50 μL of a cocktail mix consisting of titrated volumes of LIVE/DEAD violet dye; anti-CD3, anti-CD14, and anti-CD16 Pacific Blue; and anti-CD19 PcP Cy5.5 (BD Biosciences). Following a 30-minute incubation at 4°C, cells were treated with cytofix/cytoperm (BD Biosciences) according to the manufacturer's instructions. Then, anti-IL-10 Alexa Fluor 647 (eBiosciences) was added and incubated at 4°C for 30 minutes. Lastly, cells were fixed with 1% PFA and acquired on an LSRII flow cytometer (BD Biosciences).^{13 (link)}