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
The LIVE/DEAD Fixable Red Dead Cell Stain Kit is a fluorescent dye-based reagent designed to identify dead cells in a sample. The dye reacts with free amines in the cell, producing a red fluorescent signal that can be detected using flow cytometry or fluorescence microscopy. The kit allows for the reliable identification of dead cells in a variety of applications, including cell viability analyses and cell-based assays.
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