Anti mouse cd16 32

Murine cells were incubated with a Ghost Dye Red 780 (Tonbo Biosciences) viability marker and anti-mouse CD16/32 (Tonbo Biosciences) in FACS buffer for 30 minutes on ice to block murine Fc receptors. Cells were then stained with the following fluorophore-conjugated anti-mouse monoclonal antibodies: CD45, CD3, CD4, CD8, ICOS [C398.4A], CD11c, CD11b, GR1 [RB6-8C5], F4-80, CD206 [C068C2], B7-1, MHC-I [34-1-2S & 28-8-6], MHC-II [M5/114.15.2], PD-1, Tim3, PD-L1, and B7x [HMH4-5G1] (all from Biolegend); and SPSYVYHQF/H-2L^d Alexa-647 conjugated tetramer [NIH Tetramer Core Facility]. All antibodies were stained for an additional 45 minutes on ice. Human cells were incubated with the Ghost Dye Red 780 viability marker for 30 minutes on ice and immediately stained with the following fluorophore-conjugated anti-human monoclonal antibodies: MHC-I, MHC-II, PD-L1, and B7x [MIH43] (all from Biolegend) on ice for 45 minutes.

After 72 h of co-culture, T and cancer cells were collected and pre-incubated with purified anti-mouse CD16/32 (TONBO, San Diego, USA) or Fc block (BD Biosciences, Franklin Lakes, NJ, USA) for 10 min at 20 °C before staining. The Transcription Factor Buffer Set (BD Biosciences, Franklin Lakes, NJ, USA) was used for intracellular staining. Antibodies used in flow cytometry experiments in this study are listed in Supplementary Table S1. When measuring the number of cells using polystyrene fluorospheres beads, a pre-determined number of beads was added to each pre-stained sample collected from the culture medium, and the number of cells after collection was calculated from the number of beads obtained using flow cytometry. CD45-positive cells were defined as T cells and all others as cancer cells. Flow cytometry was performed using a BD LSFortessa coupled with FACSDiva v6.1.3 and data analysed using FlowJo v10.7.1 software (all from BD Biosciences).