Zombie nir fixable viability stain

0.5 to 1.0 × 10⁶ cells from each sample were aliquoted into 5ml round bottom tubes or 96-well U-bottom plates for antibody staining. Cells were washed with PBS and stained for viability using Zombie NIR Fixable Viability stain (1:2500, BioLegend 423106) in PBS for 20 min at room temperature in dark. Cells were washed with FACS buffer, blocked with Human TrueStain FcX (1:200, Biolegend 422302), and stained with the surface antibody cocktail in FACS buffer for 30 min at 4C in dark. All antibodies and dilutions used for staining are listed in Table S1 and Table S2.
For intracellular staining, cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience 00-5523-00). To block non-specific binding, 10ul of FBS was added to the cells in the residual volume of perm buffer and incubated for 5 min. Without washing, the intracellular antibody cocktail was added to the cells and stained for overnight at 4C. After the staining, the cells were washed twice with perm buffer, once with FACS buffer, and resuspended in FACS buffer for analysis. Antibody-stained cells were analyzed on an Aurora (Cytek) and the data analysis was performed using FlowJo (BD). Dimensionality reduction and hierarchical clustering analyses were performed using R functions and packages including ComplexHeatmap.

Thawed cells were stained with Zombie NIR Fixable Viability stain in PBS (BioLegend, cat #423105), followed by staining with cell-surface antibodies (see Supplementary Table S6). Stained cells were fixed and permeabilized using the Fixation/Permeabilization Solution Kit (BD Biosciences, cat #554714) according to the manufacturer's instructions, followed by a secondary permeabilization step for enhanced intracellular staining using Permeabilization Buffer Plus (BD Biosciences, cat #561651). Fixed and permeabilized cells were stained separately for anti-human-BCL2-PE (clone 124, Cell Signaling Technology, cat #26295S), anti-human-MCL1-PE (clone D2W9E, Cell Signaling Technology, cat #65617S), and anti-human-BCL-xL-PE (clone 54H6, Cell Signaling Technology, cat #13835S), or together for anti-human-BCL2-AF647 (clone 124, Cell Signaling Technology, cat #82655), anti-human-MCL1-AF488 (clone D2W9E, Cell Signaling Technology, cat #58326) and anti-human-BCL-xL-PE (clone 54H6, Cell Signaling Technology, cat #13835S). Samples were analyzed with BD LSRFortessa Cell Analyzer.