Anti mouse gr 1

Cells were collected from bronchoalveolar lavage fluid (BALF) and pre-incubated with Fc-blocking anti-mouse CD16/32 antibody (BD Pharmingen, #553,141) before the staining process. Subsequently, dead cells were excluded by DAPI staining (BD Pharmingen, #564,907). The cells then underwent surface staining with anti-mouse CD45 (BD Pharmingen, #561,037), anti-mouse CD11b (BD Pharmingen, #561,688), anti-mouse GR-1 (BD Pharmingen, #561,103), anti-mouse CD-11c (BD Pharmingen, #561,022), and anti-mouse MHCII (Biolegend, #107,613) for 30 min at 4 °C. All samples were analyzed using Cytek Dxp Athena flow cytometer, and the data were processed using FlowJo software (version 10). The method for eosinophil count was conducted following established protocols from previous study [23 (link)], and is illustrated in Fig. S1. Initially, live leukocytes were selected based on the expression of CD45 and exclusion of DAPI, effectively removing debris, erythrocytes, and dead cells. Subsequently, lymphocytes were differentiated through the analysis of the SSC-A/CD11b plot, while neutrophils were identified using the SSC-A/GR1 plot among the diverse cell populations. Ultimately, eosinophils were isolated from the remaining cells using the MHC-II/CD11c plot.

FITC anti-mouse CD8a, PE or APC anti-mouse CD4, FITC or Percp-Cy 5.5 anti-mouse CD11b, PE anti-mouse CD11c, PE or APC anti-mouse CD69, PE anti-mouse F4/80, PE anti-mouse Foxp3, FITC or PE anti-mouse IFN-γ, FITC or PE anti-mouse Ly-G and Ly-6C, anti-mouse Gr1, and isotype-matched mAbs were purchased from BD Biosciences; anti-JNK antibody and anti-p-JNK antibody were purchased from Cell Signaling Tecnology. Anti-mouse A20 antibody was purchased from Abcam; Alexa Fluor 488-conjugated donkey-anti-rabbit secondary antibody was purchased from Invitrogen. Fixation/Permeabilization Kit was purchased from eBioscience. OT-I peptide and OT-II peptide were purchased from Invivogen. For cell surface staining, cells were directly stained with either IgG control or fluorescence-conjugated antibodies; for intracellular cytokine staining, cells were permeabilized and fixed after surface staining, and stained with fluorescence-conjugated antibodies or IgG controls. Lymph nodes used in experiments were near the tumors. Analysis was carried out on a FACS calibur flow cytometer (BD Biosciences). CD11b positive cell sorting was performed by Aria I (BD Biosciences). Annexin-V-FLOUS Staining Kit was purchased from Roche and cell apoptosis was determined according to the manufacturer’s protocol.

BAL was performed as previously described22 (link). The immunomagnetic separation system (BD Biosciences Pharmingen, San Diego, CA) was used to isolate AM from BAL fluid. Magnetic nanoparticle-conjugated antibodies (anti-mouse Gr-1, anti-CD4, anti-CD8, and anti-CD45R/B220 antibodies; BD Biosciences Pharmingen, San Diego, CA) were chosen to label and remove PMN and lymphocytes. The resulting cells consisted of >98% macrophages, and cell viability was >95%.