Mhcii af700

Anti-mouse CD11c-FITC, CD8-PE-Cy7, TCRβ-PE-Cy5.5, CD11b-Pacific Blue, B220-PE-Cy7, CD4-Pacific Blue, Foxp3-APC and CD86-PE antibodies were purchased from BD Biosciences. MHCII-AF700 and CD11c-Pacific Blue were purchased from Biolegend. 2×10⁶ cells were stained in PBS containing 5% fetal calf serum (FCS) and 0.1% w/v sodium azide at 4°C for 30 min, washed with the same buffer and fixed with 1% paraformaldehyde containing 2 mM EDTA. Data were acquired on 4-laser LSR II using FACSDiva software (Becton Dickinson) and analyzed using Flow Jo 9.0 software (Tree Star, OR). For splenic DC subset analyses, TCRβ- CD11c+ cells were gated on and the MHCII and CD86 expression levels evaluated in CD11c+CD8+ and CD11c+CD11b+ cells.

100 µL of the above cell suspensions was transferred to a round bottom 96-well plate and centrifuged (3000 rpm, 1 min, 4 °C). Cell were blocked at 4 °C for 15 min using 24G2 blocking antibody (Preclin Biosystems AG) at 800 µg/mL final concentration. Cells were washed by centrifugation and resuspension with PBS/0.2% BSA. Cell were then centrifuged (3000 rpm, 1 min, 4 °C) and resuspended in 50 µl of staining solution containing appropriate primary antibodies (CD11b-PerCp-Cy5.5; CD11c-APC-Cy7; MHCII-AF700; F4/80-AF647; CD40-PE/Cy5; CD86-PE; CD80-PE/Cy7; CD4-Pacific blue; CD8-APC-Cy7; CD25-AF700; CD44-PE; all Biolegend) for 30 min at 4 °C. After two washes with PBS/0.2% BSA and one wash in PBS cells were fixed in 50 µL of BD FACS lysing solution for 15 min at 4 °C. Cells were then washed twice in PBS/0.2% BSA and resuspended in 100 µL of PBS/0.2% BSA. For intracellular staining cells were washed once more in 0.5% saponin. Cells were then incubated with primary antibody (FoxP3-AF647, Biolegend) overnight at 4 °C in 0.5% saponin. Cells were then washed once with 0.5% saponin and twice with PBS/0.2% BSA before being resuspended in 100 µL of PBS/0.2% BSA for flow cytometry analysis.