Ghost dye red 780

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.

Fc receptor blocking was performed for B cells or BLCLs. The cells were stained with antibodies in the dark. Before TCL1A (eBioscience cat# 12-6699-42) or KI67 (Biolegend cat# 350,513) staining, Ghost Dye Red 780 staining was performed, followed by staining for surface markers for 30 min at 4 °C in the dark. The cell membranes and nuclear membranes were permeabilized using a kit (Tonbo Biosciences cat# TNB-0607-KIT) prior to TCL1A or KI67 staining. A DxFLEX instrument (Backman) was used to perform flow cytometry, and the data were analyzed using FlowJo 10.5.3 software. In addition, the fluorescence minus one method was used to establish gates.