Fitc conjugated anti rat cd86

For fluorescence-activated cell sorting (FACS) analysis, 30 μg of statin-Dex or control-Dex (or 30 μg FCS protein for negative control) were incubated with 10 μl of 4 μm-diameter aldehyde/sulfate latex beads (Invitrogen, Eugene, OR, USA) for 15 min at room temperature, followed by 2 h of gentle shaking in PBS. The reaction was stopped by incubation for 30 min in 100 mM glycine. Dex- or FCS-coated beads were washed three times in FACS wash buffer (3 % FCS and 0.1 % NaN₃ in PBS) and re-suspended in 500 μl FACS wash buffer. Then, 10 μl of coated beads were incubated with phycoerythrin (PE)-conjugated anti-rat CD80 (BioLegend, San Diego, CA, USA), fluorescein isothiocyanate (FITC)-conjugated anti-rat CD86 (BioLegend), FITC-conjugated anti-rat MHC class II (eBioscience, San Diego, CA, USA), and FITC-conjugated anti-rat FasL antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by washing and analysis using an FACScan (Beckman Coulter, Los Angeles, CA, USA).

For phenotypic analysis, DCs were washed with 0.5 % bovine serum albumin (BSA; Sigma-Aldrich) in PBS and stained with PE-conjugated anti-rat CD80 (BioLegend, San Diego, CA, USA), FITC-conjugated anti-rat CD86 (BioLegend), and FITC-conjugated anti-rat MHC class II monoclonal antibodies (eBioscience, San Die go, CA, USA) for 30 min at 4 °C in the dark, respectively.