Neutrophil isolation kit

To confirm the presence of neutrophils in the human entheseal tissue, cells released following enthesis digestion (n=3), described above, were incubated with 10% mouse serum and 1% IgG in FACS buffer prior to incubation with antibodies. Cells were then stained with DAPI (live/dead), CD45+ (leukocytes), CD66b+ (granulocytes) and CD16+ (neutrophils). Cells were analyzed using the FACSMELODY (BD Biosciences) and FlowJo software (Tree Star Software, San Carlos, California, USA). All lists of antibodies, clones and manufacturers can be found in the supplementary material (Supplementary Table 1). For functional studies, neutrophils were isolated from digested entheseal tissue using a neutrophil isolation kit (STEMCELL Technologies, Cambridge, MA). This kit was validated for neutrophil purity prior to proceeding with fungal adjuvant stimulation. After 48 h of culture, isolated neutrophils evaluated through flow cytometry show 94% purity for CD66b. Isolated entheseal neutrophils were subsequently cultured in RPMI (Sigma-Aldrich) supplemented with 10% FCS and 1% penicillin/streptomycin (Invitrogen). 5x10⁵ neutrophils were plated in 96-well plates and stimulated with Zymosan (0.5mg/ml) (Sigma-Aldrich), IFNγ (20 ng/ml) (Peprotech) or Zymosan plus IFNγ for 48 h (n=4). The supernatant was then probed for IL-23 secretion by ELISA (ThermoFisher) according to the manufacturer’s instructions.

Neutrophils are enriched to 82.7% purity (Supplementary Fig S1E) using a neutrophil isolation kit according to manufacturer's instructions (Stem Cell Technology) and incubated in 180 μl murine fracture supernatant + 10% FCS + 1% penicillin/streptomycin at 37°C, 4% CO₂, and a density of 275,000 cells per well in a 96-well plate. rhTNF or PBS (in 20 μl) is added at 30 min. Cells and supernatants are harvested at 1 h. Protein levels of mCCL2 cytokine in the supernatants were analyzed as described above. Experiments were performed in triplicates.

Neutrophil isolation kit (17957, STEMCELL, Vancouver, Canada); Bovine serum albumin (A1933-25G, Sigma-Aldrich, St. Louis, MO, USA), FBS (iCell-0500, iCell Bioscience Inc, Shanghai, China); RPMI 1640 (SH30809.01, Hyclone, Logan, UT, USA); Fluorescein (FITC-dextran, 10 kDa; final concentration 5 μM, Sigma-Aldrich, St. Louis, MO, USA); fMLP (F3506, 1 mM, Sigma-Aldrich, St. Louis, MO, USA); Triton X-100 (9036-19-5, Sigma, St. Louis, MO, MO, USA); Giemsa (G5637, Sigma, St. Louis, MO, USA); D-glucose (G7021, Sigma-Aldrich, St. Louis, MO, USA); mannitol (10 mM, an osmotic control reagent, M1902, Sigma-Aldrich, St. Louis, MO, USA); Fibronectin (F2006, 2 μg mL⁻¹, Sigma-Aldrich, St. Louis, MO, USA); Commercial magnetic Neutrophil isolation kit (17957, STEMCELL Technologies, Inc., Toronto, Canada); Precursors and initiators of Polydimethylsiloxane (PDMS) (Dow Corning, Midland, MI, USA); Glass slide (80350-0001, Jiangsu Shitai Xinchuang Scientific Instrument Co., Ltd., Nanjing, China); Microscope (DMi8, Leica, Wezler, Hesse, Germany); Plasma cleaner (PDC-002, Harrick Scientific Products, Inc., New York, NY, USA).