Anti mouse il 10 apc

Dissected nerves were mechanically dissociated with a cell strainer (70 μm pores), and cells were collected in Petri dishes with cold PBS. To increase intracellular protein production, cells were treated for 3 h at 37°C with Phorbol 12-myristate 13-acetate (PMA, 50 ηg/mL, Sigma) and Ionomycin (500 ηg/mL). Brefeldin A (1 μg/mL) was added to prevent protein release by the cells. Samples were fixed and permeabilized with commercial buffers (Fixation/Permeabilization buffers, eBioscience, Cat. code: 00–5521) according to the manufacturer’s instructions, then washed with PBS, and incubated with antibodies as follows: anti-CD8/FITC, anti-IL-10/APC and anti-IFN-g/PE. 30,000 events were acquired from each sample in the flow cytometer (FACS Canto, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed in FlowJo 10.0.5 software (Tree Star Inc., Ashland, OR, USA). Gating for flow cytometry was performed using unstained cells and FMO (Fluorescence Minus One) analysis, as described elsewhere [16 (link)]. For cell staining, we used these antibodies: anti-mouse CD8a FITC (0.5 μg, eBioscience, Cat. code: 11–0081), anti-mouse IFN-γ PE (0.5 μg, eBioscience, Cat. code: 12–7311) and anti-mouse IL-10 APC (0.5 μg, eBioscience, Cat. code: 17–7101).