Dendritic cell viability and maturation resistance.

DC viability was inspected through fluorescence microscopy imaging via Zeiss AxioVision 200M using LIVE/DEAD imaging stain kit (ThermoFisher Scientific, Waltham, VA), and quantified via flow cytometry using LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit (ThermoFisher Scientific, Waltham, VA), according to manufacturer’s instructions. DC maturation was evaluated by the surface expression of stimulatory and co-stimulatory markers (MHC II, CD80 and CD86) as well as cytokine release (IL-10 and IL-12p70) by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. Following IDO incubation, DCs were challenged with 1 μg/mL of lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO) and supernatant was collected and stored at −20°C for subsequent analysis using BD OptEIA ELISA kits (BD Biosciences, Franklin Lakes, NJ) following manufacturer’s instructions. Adherent cells were incubated with 5 mM Na₂EDTA in PBS solution at 37°C for 30 minutes and lifted using a cell scraper. Cells were then washed with 1% fetal bovine serum in PBS and incubated with viability dyes mentioned above, followed by washing and incubation with antibodies (BD Bioscience, San Jose, CA) against CD16/32 (Fcγ III/II receptor) (clone 2.4G2) for 30 mins on ice to block Fcγ receptors on DCs. Cells were washed and stained with antibodies against CD11c (clone HL3) CD80 (clone 16-10A1), CD86 (clone GL1) and MHC II (clone M5/114.15.2).