Apc cy7 conjugated anti cd11c

Isotype control antibodies (Abs) (IgG1, IgG2a and IgG2b), Alexa647-conjugated anti-BDCA1 (IgG1, L161), PerCP/Cy5.5-conjugated anti-BDCA3 (IgG1, M80), APC/Cy7-conjugated anti-CD11c (IgG1, 3.9), Brilliant Violet510-conjugated anti-CD80 (IgG1, 2D10), PE-conjugated anti-CD83 (IgG1, HB15e), PE/Cy7-conjugated anti-CD86 (IgG2b, IT2.2), PE-conjugated HLA-DR, DP, DQ (IgG2a, Tu39) and PE/Cy7-conjugated HLA-A, B, C (IgG2a, W6/32) were obtained from BioLegend (San Diego, CA, USA). Antibodies against phosphorylated forms of p38, JNK and ERK were obtained from Cell Signaling Technology (Beverly, MA, USA).

Cell staining was performed at 4 °C for 20 min after Fc-blocking (anti-CD16/32 clone 93 antibody, ×200, Biolegend, San Diego, CA, United States) blocking for 15 min with the following monoclonal antibodies: FITC-conjugated anti-CD45 ( × 200, clone: 30-F11) (Biolegend), PE-conjugated anti-CD11b (×200, clone: M1/70) (Biolegend), APC-conjugated anti-CD206 (MMR) (×20, clone: C068C2) (Biolegend), APC/Cy7-conjugated anti-CD11c (×20, clone: MGL1/MGL2) (Biolegend), biotinylated anti-F4/80 (×100, clone: BM8) (Biolegend), and streptavidin PE-Cy7 conjugate (×100) for 20 min. Then the propidium iodide (PI, PerCP/Cy5.5 conjugate, 20 μg/ml) was used to define dead cells. Fluorescent levels were measured by FACS Verse (BD Biosciences) with 200000 cells. All data were analyzed with FlowJo software (BD Bioscience, version 10).