Live dead fixable red

For the human cell lines, FACS analysis was performed as follows: Cells were scraped upon reaching up to 90% confluence. Cell pellets were then washed and incubated with HLA antibodies at a 1:10 ratio in FACS buffer containing 1% FBS, 0.1% sodium azide in PBS. Measurements were performed using the Gallios flow cytometer (Beckman Coulter). Three biological replicates were analyzed, each with technical triplicates.
For the YUMMER 1.7 D4J cell line analysis the protocol was performed as follows: Acat1-KO and control D4J cells were grown to 70% confluence and scraped using ice-cold PBS. Cell pellets were stained with Live/Dead fixable Red (Invitrogen) followed by surface staining with H2-kb/H2-kd and Pdl1 in FACS buffer (PBS containing 1% FBS, 0.1% sodium azide). For digested tumor samples, single cell suspensions were stained with Live/Dead fixable Red (Thermo Fisher Scientific) in PBS followed by surface staining with antibodies in FACS buffer. For intracellular staining, single cell suspensions were stimulated with PMA (50ng)/Ionomycin (500ng) in the presence of Brefeldin A (BD Biosciences) for 4 h. After fixation and permeabilization, following manufacturer’s recommendations, cells were stained for intracellular cytokines. All samples were acquired on a BD LSRII flow cytometer and analyzed with FlowJo software.

LIVE/DEAD Fixable Red or LIVE/DEAD Fixable Aqua (Invitrogen) staining was performed at 4°C or on ice for 15 min in PBS. Surface Ab staining was performed at 4°C or on ice for 20 min in PBS containing 2% FBS and 2 mM EDTA. For staining intracellular transcription factors, fixation and permeabilization was performed with the eBioscience Intracellular Fixation & Permeabilization Buffer Set Kit prior to intracellular staining for 1 h at 4°C or on ice. Abs for flow cytometry included CD4 (RM4-5; Invitrogen), CD8 (53-6.7; eBioscience), CD25 (PC61; BD Biosciences), CD44 (IM7; eBioscience), CD62L (MEL-14; BD Biosciences), and Foxp3 (FJK-16S; eBioscience). Cells were analyzed or sorted on LSR II, LSRFortessa, and FACSAria II flow cytometers (BD Biosciences) with BD FACSDiva software. Data analysis was performed using FlowJo v.10.2 (Tree Star). FACS data presented in Fig. 3 are gated on forward scatter and side scatter properties indicative of singlet lymphocytes and live CD4⁺CD8⁻(CD4SP) cells.