Intracellular staining kit for foxp3 and ifn γ

Collected tumor tissues were minced and incubated in RPMI 1640 medium containing 2% FBS, 1mg/mL collagenase IV (Sigma: #C5138), 0.1mg hyaluronidase (Sigma: #H6254), and 200U DNase I (Sigma: #D5025) at 37°C for 1 hours to make single cells. In vitro virus-infected cells or single cells from tumor tissues were blocked with α-CD16/32 Ab (clone 93, eBioscience: #14-0161-85; 1:1000) and then cells were stained with 100 μL Zombie Aqua Fixable Viability Kit cell dye (BioLegend, San Diego, CA, USA) at a dilution of 1:1000 and left in room temperature for 15 min in the dark. Cells were stained in 100 μL total stain volume (50 μL BV stain buffer, 50 μl 2% FBS) with antibody at a dilution of 1:200 for 30 min on ice in the dark. The sources of antibodies are listed in Supplementary Table 1. The intracellular staining kit for Foxp3 and IFN-γ was purchased from BioLegend. Cells were then washed once with FACS buffer and fixed in 1% paraformaldehyde (EK Industries, Joliet, IL, USA) before being stored at 4°C overnight and acquired the next day on a BD LSR Fortessa II analyzer (BD Biosciences, San Jose, CA, USA). Data were analyzed using flowJo cytometer software.