Comprehensive Immune Cell Profiling in Murine Tissues

Peritoneal exudate cell (PEC) suspensions were blocked with Fc Block (BD Biosciences, San Jose, CA, USA) and subsequently stained with specific Abs including: anti-Ly6G FITC, anti-CD11cPE, anti-CD11b PerCP/CY5.5, Siglec-F PE, anti-c-kit APC, F4/80 APC, anti-CD4APC, anti-CD19 PerCP/Cy5.5, CD3PE (BD Biosciences, San Jose, CA, USA) and anti-CD206PE or anti-CD206 Alexa fluor 488 (Biolegend, USA). Cells were collected 4 hours after microparticle inoculation for analysis of phosphorylation of BTK (Y-551) or SYK (Y-348). Intracellular staining was performed using anti-mouse phospho-BTK/ITK (Y551/Y511) PE (eBiosciences, San Diego, CA, USA), anti-mouse phospho-SYK (Y-348) PE antibody (BD Biosciences, San Jose, CA, USA) and FOXP3/Transcription Factor Staining Buffer Set, which was used as per the manufacturer instructions (eBiosciences, San Diego,CA, USA). For CFSE-labeled cells, anti-CD4 PerCP (BD Biosciences, San Jose, CA, USA), and KJ1–26 PE (BD Biosciences, San Jose, CA, USA) were used to phenotype DO11.10 T cells and cell cycle progression was examined as previously discussed^{10 (link)}. For FACS analysis of whole synovial tissues, samples were digested at 37°C for 1 hour with 0.1% collagenase in RPMI supplemented with 10% FBS, 1000U/ml penicillin, 1000U/ml streptomycin, and 2nm L-glutamine. Single cell suspensions were then prepared and blocked and stained as described above.