Anti human cd69 pe

For the T cell activation assay, CD3+ T cells or PBMCs were co-cultured with tumor cells and 1 μg/mL of αB7-H3/CD3 or other antibodies (anti-CD3 mAb or anti-B7-H3 mAb or mixture) for 24 h in 96-well culture plates (E:T = 10:1). After that, cells were collected and stained with detection antibodies. Part of cells were stained with APC-anti-human CD4 (357408, BioLegend, San Diego, CA, USA), PC5.5-anti-human CD8 (344710, BioLegend, San Diego, CA, USA), and PE-anti-human CD69 (310906, BioLegend, San Diego, CA, USA) for 30 min in the dark. Another portion was stained with APC-anti-human CD4 (357408, BioLegend, San Diego, CA, USA) and PC5.5-anti-human CD8 (344710, BioLegend, San Diego, CA, USA), and then fixed and permeabilized prior to staining with PE-anti-human/mouse granule granzyme B (GrB) (372208, BioLegend, San Diego, CA, USA). T cell activation was determined by flow cytometry. Supernatants were collected to quantify the release of interleukin 2 (IL-2) (431804, BioLegend, San Diego, CA, USA), interleukin 6 (IL-6) (430504, BioLegend, San Diego, CA, USA), and IFN-γ (430104, BioLegend, San Diego, CA, USA) after cell culture for 48 h using ELISA kits.

To detect the activation of the Jurkat cell line, we used anti-human CD69 PE (BioLegend). To analyze the phenotype of mouse eosinophils in BALF or lung tissues, we used anti-mouse CD45 PE-CY7 (BioLegend), anti-mouse SiglecF PE (BD), anti-mouse F4/80 APC-CY7 (BioLegend), anti-mouse CD11b FITC (BioLegend) and anti-mouse CD11c APC (BioLegend). To detect mIL-5Rα positive cells, we used anti-mouse CD125 AF488 (BD). To detect mIL-5-anchored CCAR and hIL-5-anchored CCAR, we used anti-mouse/human IL-5 PE (BioLegend) or anti-HA.11 Epitope Tag AF647 (BioLegend). To detect anti-hIL-5Rα CAR, hCCL11-anchored CCAR, and hCCL24-anchored CCAR, we used anti-HA.11 Epitope Tag AF647 (BioLegend).