Siglecf pe

Whole-lung, single-cell suspensions were stained with CD11c-BV421 (Biolegend, N418) and Siglec-F-PE (BD Biosciences, E50-2440) and CD11c⁺Siglec-F⁺ cells were sorted directly into TRIzol LS Reagent (Ambion) with the BD FACSAria II (BD Biosciences). For whole-lung phenotyping, whole-lung, single-cell suspensions were stained with CD3-AF488 (Biolegend, 17A2), CD11c-BV421 (Biolegend, N418), CD8a-BV650 (Biolegend 53-6.7), CD4-BV786 (BD Biosciences GK1.5), SiglecF-PE (BD Biosciences, E50-2440), and Zombie NIR (Biolegend). Intracellular Ki-67-FITC (Biolegend, 11F6) staining was performed with the eBioscience Transcription Factor Staining Buffer Set (ThermoFisher) according to the manufacturer’s instructions. Annexin V staining was performed with PerCP-Cy5-labeled Annexin V (Biolegend) in Annexin V Binding Buffer (Biolegend) according to the manufacturer’s instructions. Data were collected with an Aurora (Cytek) or LSRFortessa (BD) and analyzed with FlowJo software.

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.