Dna intercalator

Cryopreserved PBMCs were thawed in a water bath pre-warmed to 37°C in 5 min. 3 × 10⁶ cells were then transferred into CSB for antibody staining. Mass cytometry antibody staining was implemented following previous study (24 (link)). The staining panel is listed in Supplementary Table 2. Stained cells were washed twice with 2 mL CSB, then incubated overnight at 4°C with 1 mL DNA Intercalator (Fluidigm, CA, USA), which was diluted in a Fix and Perm Buffer (Fluidigm) at a final concentration of 125 nM, which facilitated the discrimination between singlets, doublets, and triplets. Prior to data acquisition, cell pellets were resuspended in distilled water containing 10% EQ Four Element Calibration Beads (Fluidigm), and the cells were filtered through a 35-μm membrane. The cells were analyzed with Helios (Fluidigm) through several runs at an acquisition rate of ~500 events per second (25 (link)). Following the manufacturer's instructions, settings were on default.

A panel of metal-conjugated antibodies was used, including an anti-camelid variable domain of heavy chain of heavy-chain (VHH) antibody for tracing CAR T cells. Metal-coated antibodies were prepared using the Maxpar X8 Antibody Labeling Kit (Fluidigm) according to the manufacturer’s instructions.
Sample preparation, antibody staining, data acquisition, readout processing were previously described [9 (link)]. In brief, cryopreserved samples were rapidly thawed and assayed for cell number and viability. Cells were stained with Cisplatin (Fluidigm), resuspended in Cell Staining Buffer (Fluidigm), and incubated with Human TruStain FcX (Biolegend). Each sample was subsequently labeled with cell surface antibody cocktail. After washing, samples were fixed in paraformaldehyde followed by treatment with the DNA intercalator (Fluidigm). The stained cells were resuspended in freshly prepared Cell Acquisition Solution (Fluidigm). Data were subsequently acquired on CyTOF in National Research Center for Translational Medicine (Shanghai). Data of individual sample were manually gated using Cytobank for downstream analysis. t-SNE and PhenoGraph algorithm were performed on all samples. Data were displayed using the ggplot2 R package.