Apc conjugated anti mouse foxp3

For cell surface molecules staining, DCs (1 x 10⁶ cells/well) were harvested, washed with FACS washing buffer (0.5% FBS and filtered 0.05% NaN₃ in PBS). The cells were then labeled for 20 min at room temperature with APC-conjugated anti-mouse CD11c (N418) and one of the following PE-conjugated mAbs: anti-mouse IA^b (AF6-120.1; MHC II), anti-mouse CD40 (3/23), anti-mouse CD80 (16-10A1) and anti-mouse CD86 (GL1) The mean fluorescence intensity (MFI) of each surface molecules was measured from the total cell population via BD accuri C6 plus flow cytometer (BD Biosciences). For intracellular staining, cells were stimulated for 5 h with 50 ng/ml of PMA, 1 μg/ml of ionomycin and Golgiplug. The cells were harvested, washed with FACS buffer and stained with FITC-conjugated anti-mouse CD4 (RM4-5) for 20 min at room temperature. The cells were fixed for 20 min using a Cytocfix/Cytoperm kit (BD Biosciences). Intracellular cytokines were detected with APC-conjugated anti-mouse IFN-γ (XMG1.2) and IL-17 (eBio17B7), PE-conjugated anti-mouse IL-4 (11B11). Treg cells were firstly stained with FITC-conjugated anti-mouse CD4 (RM4-5) and PE-conjugated anti-mouse CD25 (PC61), followed by APC-conjugated anti-mouse FoxP3 (FJK-16s) according to the manufacturer’s instructions of FoxP3 staining buffer set (eBioscience, San Jose, CA).

APC conjugated anti-mouse CD4 (17-0041-83), PB conjugated anti-mouse CD4 (48-0042-82), PE-cy7 conjugated anti-mouse CD8 (25-0081-82), APC-cy7 conjugated anti-mouse CD11b (47-0112-82), PB conjugated anti-mouse CD11c (48-0114-82), PerCP conjugated anti-mouse Ly6G (46-9668-82), FITC conjugated anti-mouse B220 (11-0452-85), PE conjugated anti-mouse CD45 (12-0451-83), APC conjugated anti-mouse IFNγ (17-7311-82), PE conjugated anti-mouse TNFα (12-7321-82), PE-cy7 conjugated anti-mouse CD25 (25-0251-82), APC conjugated anti-mouse Foxp3 (17-5773-82), PE conjugated anti-mouse CD44 (12-0441-83), FITC conjugated anti-mouse CD62L (11-0621-86), anti-mouse CD3 (16-0031-86), and anti-mouse CD28 (16-0281-86) antibodies were purchased from eBioscience. BrdU Flow Kits (559619) were purchased from BD Biosciences. Anti-mouse Ly6G (BE0075) antibody was purchased from Bioxcell. Alexa Fluor 488-conjugated anti-Rat IgG secondary antibody (A-21210) was from Thermo Fisher. Mouse anti-CD4 (L3T4, 130-049-201) and mouse anti-Ly-6G (130-092-332) Micro Beads were purchased from Miltenyi Biotec. Murine IL-25 (1399) were purchased from R&D. Lipopolysaccharides (LPS, L3129) were purchased from Sigma. P. acnes were prepared as previously described (7 (link)).

The following monoclonal antibodies were used during the assay: Brilliant violet (BV)510-conjugated anti-mouse CD3 (BD-Biosciences), BV510-conjugated anti-mouse γ/δTCR (BD-Biosciences) recognizing delta chain, fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD4 (BD-Biosciences), FITC-conjugated anti-mouse CD107a (BD-Biosciences), FITC-conjugated anti-mouse CD49b (BD-Biosciences), phycoerythrin (PE)-conjugated anti-mouse TIM-3 (R&D Systems), PE-conjugated anti-mouse Gal-9 (Biolegend), PE-conjugated anti-mouse CD49b (BD-Biosciences), peridinin chlorophyll a protein (PerCP)-conjugated anti-mouse CD45 (Exbio), PE-Cy7-conjugated anti-mouse CD25 (BD-Biosciences), allophycocyanin (APC)-conjugated anti-mouse TIM-3 (R&D Systems), APC-conjugated anti-mouse FoxP3 (eBioscience) and APC-H7-conjugated anti-mouse CD8 (BD-Biosciences). Control antibodies included isotype-matched rat and hamster antibodies.