Anti ly6g percp

Cells from lymph nodes (5x10⁵) or ear homogenates (1x10⁶) were washed with PBS at 400 g for 5 min at 4°C and blocked with Human FcX (BioLegend) for 15 min, followed by staining with the antibody cocktail for 30 min at 4°C. Cells were then washed with a cytometry buffer (PBS with 5% FBS) at 400 g for 5 min and 4°C), then fixed with 4% formaldehyde (Sigma) for 15 min at 4°C. Cells were washed and resuspended in the cytometry buffer and stored in the dark at 4°C until acquisition. The following antibodies were used: anti-PD-L1-APC, anti-CD10-APC-780 (human, eBioscience); anti-CD45-APCcy7, anti-CD11b-FITC, anti-CD11b-PE, anti-CD11b-PEcy7, anti-Ly6G-PerCP, anti-Ly6G-FITC and anti-PD-L1-APC (murine, eBioscience). Acquisition of events (lymph node, 100,000 events; ear, all cells) was performed on a BD FACSAria™. The gate strategy was performed based on the selection of cell size (FSC) and composition (SSC). After identifying the main population, a gate of FSC-A (area) and FSC-H (weight) was used, where cellular doublets were excluded. Gates for positive events were established through Fluorescence Minus One (FMO) control. The data analyzes were performed using the FlowJo software.