Anti cd11b pacific blue

For direct immunofluorescence labeling, macrophages were stained with the following fluorescence-labeled anti-human mAbs: anti-CD40-FITC, anti-CD64-A700, anti-CD80-V450 and anti-CD86-APC, anti-CD11b-pacific blue, anti-CD163-PE, anti-CD206-FITC (all from BD Biosciences, San Diego, CA) and anti-CD200R-APC Abs (Serotec, Oxford, UK). For Notch1 receptor analysis, macrophages were immunostained using rabbit anti-Notch1 (1:100 dilution, ab52627 from Abcam, Cambridge, UK) Abs and Alexa-488 labeled anti-rabbit IgG as secondary antibodies. For apoptosis analysis, cells were incubated with Annexin V/propidium iodide following the manufacturer’s recommendations (Life Technologies). Cells stained with, isotype-matched, irrelevant Abs were used as negative controls. Fluorescence was measured by flow cytometry after gating on FSC/SSC parameters using a FACS LSR II® (BD Biosciences) and next analyzed using FlowJo® VX software (Tree Star, Inc., Ashland, OR, USA).

Spleens were harvested from untreated wild-type, TNFR1 KO and SPRET mice, and single cells were obtained by teasing the spleen apart by pressing it through a 100-μm cell strainer, eliminating clumps and debris. Cells were collected in 10 ml DPBS and were spin down (4°C, 7 min, 400× g). Cells were lysed with 1 ml of lysis buffer (8.3 g NH₄Cl, 1 g KHCO₃ and 200 μl 0.5 mol/l EDTA), washed and counted. A total of 1 × 10⁶ splenocytes were stained for 30 min at 4°C in the dark with the following anti-mouse antibodies: anti-TCRb-FITC, anti-CD19-PECy7, anti-CD11b-Pacific Blue, anti-Ly6C-APC, anti-Ly6G-AF700 (all from BD Biosciences) and anti-TNFR1-PE (BioLegend). Cells were washed and dissolved in FACS buffer. Fluorescent events were acquired using an LSR2 and analysed using FACSDiva software (BD Biosciences). Neutrophils were defined as SSC^highCD11b⁺Ly6G^highLy6C^high. After gating neutrophils (see Supplementary Fig S7), TNFR1⁺ cells were plotted in a histogram. The correct neutrophil population and TNFR1⁺ populations were gated using an FMO conditions without anti-Ly6G-AF700 and anti-TNFR1-PE antibodies, respectively. Analysis was performed using FlowJo software (Tree Star, Inc.)

Spleen and inguinal lymph node (LN) cells were harvested from immunized mice at the time of sacrifice. Joint tissues were minced and treated with collagenase (0.25 mg/ml), and joint cell populations were examined for surface markers using antibodies anti-CD4-AF700, anti-CD8a-PE-Cy7, anti-CD19-PE, anti-CD11b-Pacific Blue, anti-CD11c-APC-Cy7, anti-F4/80-APC, and anti-GR-1-FITC (BD Pharmingen, San Jose, CA, USA). For intracellular cytokine staining, cells were stimulated with Phorbol 12-myristate 13-acetate (25 ng/ml) and ionomycin (500 ng/ml) (Sigma-Aldrich) and treated with GolgiPlug (BD Pharmingen) for 4 hours. After cell surface staining with anti-CD3e-PE-Cy7 and anti-CD4-APC-Cy7, cells were permeabilized using the Cytofix/Cytoperm Plus kit (BD Pharmingen) and stained with anti-IFNγ-APC and anti-IL-17A-FITC. A BD LSRII cytometer was used for cytometry and data were analyzed using BD FACS Diva Software.