Anti mouse cd16 32 clone 13

Single-cell suspensions from draining lymph nodes or tumor cell suspensions were incubated with anti-mouse CD16/32 (clone 13; BioLegend) to block Fc receptors for 10 min on ice and fixed for 20 min at room temperature, followed by washing with permeabilization buffer (Foxp3/Transcription Factor Staining Buffer Kit; TONBO). Cells were incubated with fluorescent antibodies in a permeabilization buffer for either 1 h at room temperature or overnight at 4°C. FACS Abs were purchased from BioLegend, Cell Signaling Technology, or Santa Cruz Biotechnology and included CD3 (145-2C11), Tubulin (YOL 1/34), CD8b (YTS156.7.7), PD-1 (RMP-1-30), and TCF1 (C63D9). Following staining, cells were washed with permeabilization buffer and stained in 0.01 mg/ML DAPI (Thermo Fisher) for 5 min before suspending in PBS for analysis using the Amnis ImageStreamX MkII imaging cytometer. Images were collected at 60× magnification, compensated using single stain controls from the spleen of tumor-challenged mice, and gated and processed using the IDEAS V6.2 software.