Detecting B10 and B10 Progenitor Cells

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)}