Easysep mouse pe

Gr1+ cells and CD8+ T cells were isolated from the spleens of EMT6 tumor-bearing mice using positive isolation EasySep Mouse PE, BioLegend, and MojoSort™ Mouse CD8 T Cell Isolation Kit, respectively. The Gr1+ cells (2x10⁵ cells/ml) were seeded in 24 well plates and co-cultured with anti-Bv8 or IgG control antibodies (10 µg/ml) for 24 hours. Cells were then collected and immunostained for G-MDSCs or M-MDSCs. To analyze cell viability, the cells were stained with 7AAD and propidium iodide (PI) and analyzed by flow cytometry. In some experiments, Gr1+ cells (0.5x10⁶ cells/ml) and CD8+ T cells (0.5x10⁶ cells/ml) were co-cultured in a 24-well plate in the presence of anti-Bv8 or IgG control antibodies for 24 hours. Subsequently, the cells were collected and analyzed by flow cytometry to detect different immune cell states as indicated in the figure and in Table S1. The flow cytometry gating strategy is shown in Figure S8B. In some experiments, granzyme B was evaluated in the conditioned medium obtained from the co-cultured system analyzed by ELISA (R&D systems). All experiments were performed at least in three biological replicates.

GR1⁺ and CD8⁺ T cells were isolated from the spleens of 4T1 tumor bearing mice using EasySep Mouse PE, and MojoSort™ Mouse CD8⁺ T Cell Isolation Kit (BioLegend, Cat# 480008), respectively. Ly6E^hi neutrophils were generated in-vitro as described above. The GR1⁺ cells and Ly6E^hi neutrophils (1x10⁶/ml) were cultured in serum-free medium for 24 hours to generate conditioned medium (CM). CD8⁺ T cells (0.5x10⁶/ml) were cultured with CM of GR1⁺ or Ly6E^hi neutrophils cells for 24 hours, after which the cells were washed and analyzed by flow cytometry for the evaluation of activated CD8⁺ T cells (CD8⁺/CD25⁺), effector CD8⁺ T cells (CD8⁺/CD44⁺/CD62L^-), Granzyme B⁺ and Ki67⁺ cells. In some experiments, CD8⁺ T cells (0.5x10⁶/ml) were cultured with conditioned medium of Ly6E^hi neutrophils together with neutralizing antibodies anti-IL12p40 (1μg/ml) or anti-IL23p19 (2μg/ml) (R&D systems, Cat# MAB4991 and Cat# AF1619, respectively). After 24 hours, the cells were collected, washed and analyzed by flow cytometry for the evaluation of activated CD8⁺ T cells (CD8⁺/CD25⁺). The experiments were performed using five biological repeats.

Gr1+ cells and CD8+ T cells were isolated from the spleens of EMT6 tumor-bearing mice (n=5 mice) using positive isolation EasySep Mouse PE, BioLegend, and MojoSort™ Mouse CD8 T Cell Isolation Kit, respectively. EMT6 cells were seeded in a 48-well plate (4000 cells/well) along with CD8+ T cells (0.5x10⁶ cells/ml) and Gr1+ cells (0.5x10⁶ cells/ml) obtained from each individual mouse, for 24 hours in the presence of anti-Bv8 or IgG control antibodies (10 µg/ml). Subsequently, PI (500 nM) was added to cultures in order to identify dead cells. T-cell killing effect was monitored by flow cytometry and Incucyte Zoom HD/2CLR system (Essen BioScience, Ann Arbor, MI). The flow cytometry gating strategy is shown in Figure S8C.