Anti ly6g pe cy7

Tumor tissues from C57BL/6J mice were collected and dissociated into single‐cell suspension by filtering with a 70‐μm mesh cell strainer. Red blood cells were lysed in ACK lysing buffer. Cells were resuspended in Percoll gradients and centrifuged at 400 g for 20 min to isolate lymphocytes. PMA, ionophore, and protein transport inhibitor were added into cell suspension for 5 h incubation. For lymphocyte subpopulations. cells were stained for analysis of surface markers including anti‐CD3 eFluor™ 450 (eBioscience), anti‐CD4 FITC (eBioscience), anti‐CD8 APC (eBioscience), and anti‐PD‐1 PE‐Cy7 (eBioscience), then fixed and permeabilized for anti‐IFN‐γ PE (eBioscience) measurement. For myeloid‐derived suppressor cells (MDSCs) and M2‐like macrophage subpopulations, cells were stained with anti‐CD11b eFluor™ 450 (eBioscience), anti‐Ly6G PE‐Cy7 (eBioscience), anti‐Ly6C PE (eBioscience), anti‐F4/80 APC (eBioscience), and anti‐CD206 FITC (eBioscience). Dead cells were stained with 7‐AAD.
Cultured cells in vitro were stained with PE‐conjugated HLA‐ABC, H‐2Kb, and PD‐L1 (eBioscience) monoclonal antibodies in the dark at 4°C for 1 h, then analyzed using a flow cytometer (BD Bioscience).

Whole blood samples were collected from mice by cardiac puncture with a syringe containing 1% heparin (Sigma), and erythrocytes were lysed using AcK (5 M – Gibco by Life Technologies). After lysis protocol, cells were then resuspended in a FACS buffer (PBS containing 0.1% sodium azide and 1% FBS) and stained with fluorescent-conjugated monoclonal antibodies anti-Ly6G-PECy7, anti-Ly6C-PE (both eBioscience), and anti-CD11b-FITC (BD Pharmingen). Results were collected for 10,000 events and analyzed using FlowJo software (TreeStar). In the full gate strategy, doublets, debris and dead cells were gated out. First, CD11b⁺ were gated and Ly6C^highand Ly6G⁺populations were determined in CD11b⁺ cells.Ly6G⁺ populations were excluded from the gate in the final analysis (see Supplementary Fig. S1).