E50 2440

Fluorescent-labeled antibodies for CCR3 (FAB729F, FITC, #83101) was purchased from R&D Systems, Siglec F (552126, PE, E50-2440) was purchased from BD Biosciences, for Gr-1 (108408, PE, RB6-8C5), CD45 (103134, brilliant violet 421, 30F11), Thy1.2 (140306, pacific blue, 53-2.1), Sca-1 (108142, AlexaFluor700 or 108112, APC, D7), CD3 (100308, PE or 100306, FITC, 145-2C11), CD4 (100512, PE or 100540, PerCP-Cy5.5, RM4-5), CD8 (100708, PE, 53-6.7), CD11b (101208, PE or 101206, FITC, M1/70), CD19 (115508, PE, 6D5), NK1.1 (108708, PE or 108706, FITC, PK136), B220 (103206, FITC, RA3-6B2), and IL-5 (504306, APC, TRFK5) were purchased from BioLegend, for IL-13 (12-7133-41, PE, eBio13A), GATA3 (12-9966-42, PE, TWAJ), and IL-25R (12-7361-82, PE, MUNC33) were purchased from eBioscience, for T1/ST2 (101001F, FITC or 101001B, biotin, DJ8) was purchased from MD Biosciences, and for IgE (1130-09L, PE, 23G3) was purchased from Southern Biotechnology Associates Inc. PerCP-Cy5.5-streptavidin was purchased from BioLegend (405214). Recombinant mouse IL-7 (407-ML) and IL-25 (1399-IL) were purchased from R&D Systems. Ionomycin and phorbol 12-myristate 13-acetate (PMA) were purchased from Sigma-Aldrich Japan. Recombinant human IL-33 (rhIL-33) was made by Hokudo Co., Ltd. as previously described (32 (link)).

For depletion of eosinophils, 20 µg of purified rat anti–mouse SiglecF (E50-2440; BD) or isotype rat IgG2a (BD) in 100 µl of sterile PBS was injected into the lateral tail vein. Experiments were performed 6 h after injection. For depletion of platelets, anti–platelet antibody (6A6-IgG2a; 0.2 µg/g body weight) or isotope IgG2a was injected into the lateral tail vein (both antibodies were provided by F. Nimmerjahn, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany). Experiments were performed 1 h after injection. Depletion of platelets and eosinophils was evaluated by an Advia hemocytometer or Gallios flow cytometer (Beckman Coulter), respectively, in separate experiments after the indicated time to avoid any interference with the in vivo assays by the vascular injury. For inhibition of 12/15-LO in vivo, 10 µg/g body weight baicalein or DMSO control in 200 µl PBS was injected intraperitoneal two times within 24 h. Experiments were performed 1 h after last injection.

A described previously [53 (link)], lungs were harvested, cut into small pieces and incubated for 1 hour at 37°C with a mix of DNAse I (100 μg/ml, Sigma-Aldrich) and collagenase (400 U/ml 1.6 mg/ml, Roche). Lung cells were washed and filtered through a 100 μM filter before being incubated with saturating doses of purified 2.4G2 (anti-mouse Fc receptor, ATCC) in 200 μL PBS 0.2% BSA 0.02% NaN3 (FACS buffer) for 20 minutes at 4°C to prevent antibody binding on the Fc receptor. Various fluorescent mAb combinations in FACS buffer were used to determine cell populations (Table 1). Acquisitions were done on FACScanto II cytofluorometer (Becton Dickinson) with the following mAbs from BD Biosciences: Fluorescein (FITC)-coupled HL3 (anti-CD11c), FITC-coupled 145-2C11 (anti-CD3), APC-coupled RB6-8C5 (anti-GR1), phycoerythrine (PE)-coupled RM4-5 (anti-CD4), PE-coupled E50-2440 (anti-SIGLEC-F), APC-coupled BM8 (anti-F4/80). APC-eF780-coupled M1/70 (antiCD11b) were purchased from eBiosciences and fixable viability dye Aqua (ThermoFisher) was used to gate viable cells. Gating strategies are summarized in S3 Fig.

Female mice (C57BL/6) were injected intraperitoneally with either PBS, Alum (0.3 mg/mouse), 0.3 mg pSi microparticles, MPL (50 µg/mouse) or pSi particles loaded with MPL. After 24 hr the mice were euthanized by CO₂, the peritoneal cavity was washed with PBS and the peritoneal extrudate cells (PEC) were harvested for flow cytometry analysis and/or ex vivo restimulation. For restimulation experiments, PECs were plated at 1×10⁶ cells/ml and restimulated with either CpG (5 µg/ml) or Heat-killed E. coli (10 bacteria:1 cell) for 24 hr. Supernatants were collected and IFN-γ, TNF-α, IL-12p40 and IL-10 concentrations were determined by ELISA.
For flow cytometry analysis, PECs (1×10⁶ cells/ml) were incubated for 30 min with AQUA fluorescent dye (0.5 µl) (Invitrogen) and for an additional 30 min with antibodies specific for CD11b (0.005 µg) (M1/70 : BD Pharmingen), Gr-1 (0.04 µg) (RB6-8C5 : BD Pharmingen), F4/80 (0.1 µg) (BM8 : eBioscience), ckit (0.5 µg) (2B8 : BD Pharmingen), CD11c (0.1 µg) (HL3 : BD Pharmingen), MHCcII (0.04 µg) (M5/114.15.2 : BD Pharmingen), CD19 (0.1 µg) (1D3 : BD Pharmingen), Ly6C (0.25 µg) (AL-21 : BD Pharmingen) and SiglecF (0.02 µg) (E50-2440 : BD Pharmingen). Samples were acquired using a Becton Dickinson FACSCanto flow cytometer equipped with Summit software (Dako, Colorado, USA) and the data was analysed using FlowJo^T software (Treestar, Oregon, USA).

PD-1 FITC (1:300; J43; eBioscience), NK1.1 PE (1:300; PK136; eBioscience), CD3e PE-Dazzle594 (1:300; 145-2C11; BioLegend), CD3e PE (1:200; 145-2C11; eBioscience), FoxP3 PerCP-Cy5.5 (1:200; FJK-16s; eBioscience), CD8a PE-Cy7 (1:2000; 53-6.7; eBioscience), CD8a APC (1:600; 53-6.7; eBioscience), CD8a PerCP-Cy5.5 (1:400; 53-6.7; eBioscience), TCRb AF700 (1:100; H57-597; BioLegend), CD45 BV785 (1:1000; 30-F11; BioLegend), CD4 BV711 (1:400; GK1.5; BioLegend), CD19 BV650 (1:400; 6D5; BioLegend), CD19 PE (1:500; 1D3; eBioscience), CD11b BV605 (1:1000; M1/70; BioLegend), CD11b BV510 (1:400; M1/70; BioLegend), CD11c BV605 (1:400; N418; BioLegend), Siglec-F PE (1:300; E50-2440; BD Biosciences), F4/80 APC (1:200; BM8; BioLegend), Ly-6G AF 700 (1:300; 1A8; BioLegend), MHC II BV650 (1:4000; M5/114.15.2; BioLegend), CD64 BV421 (1:200; X54-5/7.1; BioLegend), TNF FITC (1:300; MP6-XT22; BioLegend), IFNg PE-Cy7 (1: 1000; XMG1.2; BD Biosciences), Eomes PE (1:200; W17001A; BioLegend), Ki-67 BV 650 (1:200, 11F6; Biolegend), GzmB FITC (1:100; GB11; BioLegend). Gating strategy is depicted in Supplementary Fig. 6d.