Fitc conjugated anti mouse cd11c

After treatment for 24 hours, BMDCs were harvested and stained with fluorescein FITC-conjugated anti-mouse CD11c, PE-conjugated anti-mouse CD11b, PE-conjugated anti-mouse CD80, PECy5-conjugated anti-mouse CD86, and PECy5-conjugated anti-mouse major histocompatibility (MHC) class II antibodies (eBioscience). The FITC Annexin V apoptosis detection kit (BD Pharmingen) was used for cell death studies. Cells were washed with PBS and fixed with 1% paraformaldehyde (Merck). Fluorescently labeled cells were analyzed by flow cytometry (FACScan, BD).

Cells (4 × 10⁵) were incubated in 100 µl FACS buffer (PBS plus 0.1% FCS) containing fluorochrome-conjugated antibodies at a concentration of 10 µg/ml. The cells were stained with following antibodies (all from eBioscience): FITC-conjugated anti-mouse CD11c and APC-conjugated anti-mouse CD86. For intracellular cytokine staining of IL-12 p70, cells were stimulated with phorbol 12-myristate 13-acetate (50 ng/ml) and ionomycin (500 ng/ml, both from Sigma-Aldrich) for 3 h and followed by addition of brefeldin A (10 μg/ml, Sigma Aldrich) for another 4 h. The cells were then stained with APC-conjugated anti-mouse IL-12p70 (Thermo Fisher Scientific). After incubating with the Abs for 60 min at 4 °C, the cells were washed twice and resuspended in FACS buffer for flow cytometric analysis (FACSAria Fusion, BD Biosciences).

For cell surface and intracellular marker analysis, 10⁶ cells were incubated with fluorescent-conjugated antibodies in labeling solution (eBioscience, USA). The fluorescent-conjugated antibodies used in this study included PE-conjugated anti-mouse CD11c, FITC-conjugated anti-mouse CD11c, FITC-conjugated anti-mouse CD86, FITC-conjugated anti-mouse MHC-II, APC-conjugated anti-mouse CD80, PE-CY5-conjugated anti-mouse CD40, APC-conjugated anti-mouse CD4, FITC-conjugated anti-mouse IL-17A, FITC-conjugated anti-mouse Foxp3, PE-conjugated anti-mouse GATA-3, PE-conjugated anti-mouse T-bet antibodies, and FITC-conjugated anti-mouse CD103 (integrin alpha E) (all purchased from eBioscience, USA). Fluorescent-conjugated, isotype-matched, irrelevant antibodies were used to establish background fluorescein levels. Flow cytometry analysis was conducted on a FACSCalibur (BD Biosciences, USA), and FACSCalibur software (BD Biosciences) was used to analyze the flow data. DCs were gated for PE-CD11c, and then FITC-MHC-II expression and endocytic FITC-OVA levels were analyzed. MLR-lymphocytes were gated for APC-CD4, and then FITC-Foxp3 was analyzed.