Tumor tissues were minced and incubated in digestion solution containing 0.5 mg/ml collagenase V, 0.2 mg/ml hyaluronidase and 0.015 mg/ml DNase I (Sigma, St. Louis, USA) in a 37 °C water bath for 1 h.
The samples were then ltered through a 70 µm strainer before staining with uorescence antibodies. Mouse Percy5-CD45, FITC-lineage, PE-CD90.2, and APC-KLRG1 (Biolegend) were used to analyze the percentage of ILC2s in the mouse tumor tissue and human Percy5-CD45, FITC-lineage, PE-CRTH2, and APC-CD127 (Biolegend) were used to analyze the percentage of ILC2s in the human tumor tissue. Mouse CD8 cells were stained with Percy5-CD45, FITC-CD3, and PE-CD8 (Biolegend). For analyzing the percentage of MDSCs, FITC-CD11b and APC-Gr-1 (Biolegend) were used. All surface stained samples were kept at 4 °C for 30 min.
For intracellular staining of IL-9, tumor tissue single-cell suspensions were rst stimulated with 50 ng/mL phorbol myristate acetate (PMA), 1 µg/mL ionomycin, and 2 µg/mL monensin (eBioscience, San Diego, USA) for 6 h. Samples were xed, permeabilized, and stained with IL-9 antibody on a shaker at 4 °C for 45 min.
The apoptosis kit FITC-Annexin V and APC-7-AAD (Multisciences, Hangzhou, China) was used following the manufacturer's protocols to analyze the apoptosis of CT26 cells.
The samples were then ltered through a 70 µm strainer before staining with uorescence antibodies. Mouse Percy5-CD45, FITC-lineage, PE-CD90.2, and APC-KLRG1 (Biolegend) were used to analyze the percentage of ILC2s in the mouse tumor tissue and human Percy5-CD45, FITC-lineage, PE-CRTH2, and APC-CD127 (Biolegend) were used to analyze the percentage of ILC2s in the human tumor tissue. Mouse CD8 cells were stained with Percy5-CD45, FITC-CD3, and PE-CD8 (Biolegend). For analyzing the percentage of MDSCs, FITC-CD11b and APC-Gr-1 (Biolegend) were used. All surface stained samples were kept at 4 °C for 30 min.
For intracellular staining of IL-9, tumor tissue single-cell suspensions were rst stimulated with 50 ng/mL phorbol myristate acetate (PMA), 1 µg/mL ionomycin, and 2 µg/mL monensin (eBioscience, San Diego, USA) for 6 h. Samples were xed, permeabilized, and stained with IL-9 antibody on a shaker at 4 °C for 45 min.
The apoptosis kit FITC-Annexin V and APC-7-AAD (Multisciences, Hangzhou, China) was used following the manufacturer's protocols to analyze the apoptosis of CT26 cells.