Triton x 100

10⁶ neutrophils/mL of RPMI-1640 medium supplemented with 2% FBS were stuck on 0.001% poly-L-lysine-treated glass coverslips (Sigma Aldrich, St. Louis, MO, USA) and incubated 4h at 37°C in 5% CO₂ atmosphere in Multiwell Plates (Corning Incorparated. Costar^R cat. 3598) with or without stimulus. After incubation, cells were fixed with 4% paraformaldehyde overnight and blocked 2h with 10% normal mouse serum. Cells were then permeabilized with 0.02% Triton X-100 (Polysciences Inc. cat. 4605) in 1 M NaCl and incubated with primary antibodies (mouse anti-human elastase or mouse anti-human histone, both kindly donated by Dr. A. Zychlinsky, Max Planck Institute for Infection Biology, Germany) which were detected with the following secondary antibodies: Alexa Fluor^R 488 goat anti-mouse IgG (Molecular Probes, cat. A-11017) and Alexa Fluor^R 594 goat anti-mouse IgG (Molecular Probes, cat. A-11020). For DNA detection 4′,6-Diamidino-2-phenylindole-dihydrochloride (DAPI) was used. Specimens were analyzed with a confocal microscope (Olympus BX51TF, Tokyo, Japan).

Whole mount staining of organoids was performed essentially as described previously [17 (link)]. Organoids were fixed in 4% PFA for 30 min followed by treatment with 50 mM NH₄Cl (Nacalai Tesque Inc., Japan, 02423-45) for 30 min. Permeabilization was performed with 0.1% Triton X-100 (Polysciences, USA, 04605 − 250) for 30 min, and then blocking was performed with 5% bovine serum albumin (Nacalai Tesque Inc., Japan, 01863-51) for 1 h. Thereafter, organoids were incubated with primary antibodies overnight at 4 °C and then with the secondary antibody for 1 h at room temperature. Primary and secondary antibodies were used in the same concentrations described earlier. All samples were treated with Hoechst 33,342 (Tocris Bioscience, UK, 5117) for nuclear staining. Images were acquired using a TCS-SP5 confocal laser scanning microscope (Leica, Germany).

NF157, MRS2500, suramin, AR-C 118925XX, CGS15943, pertussis toxin (PTX), ATP, and ATPγS were from Tocris Bioscience (Bristol, UK). YM-254890 (YM) was from Wako Pure Chemical Industries (Richmond, VA). Bovine serum albumin (BSA) fraction V fatty acid-free was from Roche (Basel, Switzerland). Poly-d-lysine and apyrase were from Sigma-Aldrich (St. Louis, MO). Carbachol was from Abcam (Cambridge, UK). Triton X-100 was from Polysciences (Warrington, PA). RANTES was from Peprotech (Rocky Hill, NJ) and RANTES analog, PSC-RANTES was a gift from Oliver Hartley (Université de Genève). HEK293T cells were from American Type Culture Collection (ATCC) (Manassas, VA). Dulbecco’s Modified Eagle’s Medium GlutaMAX (DMEM), FluoroBrite DMEM, Dulbecco’s phosphate-buffered saline without Ca²⁺ and Mg²⁺ (DPBS), Hanks' Balanced Salt solution (HBSS), and HEPES buffer were from Fisher Scientific (Hampton, NH). Lipofectamine 2000 and trypsin–EDTA (0.25%, phenol red) were from ThermoFisher Scientific (Waltham, MA). Fetal bovine serum (FBS) was from Gemini Bio-Products (West Sacramento, CA). Clear and clear-bottom black 384-well microplates were from Greiner (Monroe, NC). FLIPR Calcium 6 Assay and Flexstation II 384 Plate Reader were from Molecular Devices (San Jose, CA). 384-well transfer tips for the Flexstation were from Axygen (Union City, CA).