Maxpar x8 multimetal labeling kit

Thawed PBMC were transferred to a 37 °C centrifuge tube containing RPMI-1640 medium and resuspended in phosphate buffered saline (PBS). PBMCs were washed with PBS on ice, stained with cisplatin (195-Pt, Fluidigm, USA), and cell viabilities were assessed. Purified antibodies were purchased from BioLegend and then conjugated with metals using the Maxpar® X8 Multimetal Labeling kit (Fluidigm) according to the manufacturer’s protocol. The list of antibodies and reporter isotopes are described in detail in Supplementary Table 1. Next, PBMCs were stained with cell-surface antibodies, and intracellular staining was performed using the Intercalator-Ir reagent (Fluidigm). Finally, the specimens were resuspended in deionized water containing 10% EQ Four Element beads (Fluidigm). A Helios mass cytometer (Fluidigm) was used for data acquisition.

Antibodies were conjugated to metal polymers using the Maxpar X8 Multimetal Labeling Kit (Fluidigm, 201300) and Ionpath Conjugation Kits (Ionpath, 600XXX) with slight modifications to manufacturer protocols. The antibodies used and their respective clones are listed in the key resources table. Antibody conjugation was performed exactly as described previously (Han et al., 2018 (link)). In short, 100ug of carrier free antibodies are subject to gentle reduction in the presence of 4uM of TCEP for 30 min, before conjugation to lanthanide-loaded polymers. Post elution, all antibodies are quantified via nanodrop (Thermo Fisher Scientific, ND2000), diluted with >30% w/v Candor PBS antibody stabilizer containing 0.02% w/v NaN3 (Thermo Fisher Scientific, nc0436689) and stored at 4°C until use.

A panel of 39 antibodies designed to distinguish a broad range of immune cells was used. Antibodies were either purchased in a preconjugated form from Fluidigm (South San Francisco, United States) or purchased from Biolegend (San Diego, United States) in a purified form and conjugated inhouse using the Maxpar X8 Multimetal Labeling Kit (Fluidigm, United States) according to the manufacturer’s instructions. The antibodies and reporter isotopes are included in Supplementary Table 1. The samples were then washed and stained with cisplatin-195Pt (Fluidigm, 201064) as a viability dye. Cell samples were then washed and stained with cell surface antibodies for 30 min on ice. Subsequently, the samples were permeabilized at 4°C overnight and stained with intracellular antibodies for 30 min on ice. The antibody-labeled samples were washed and incubated in 0.125 nM intercalator-Ir (Fluidigm, United States) diluted in phosphate-buffered saline (PBS, Sigma-Aldrich, United States) containing 2% formaldehyde and stored at 4°C until mass cytometry examination. Before acquisition, the samples were washed with deionized water and then resuspended at a concentration of 1 x 10⁶ cells/mL in deionized water containing a 1:20 dilution of EQ Four Element Beads (Fluidigm, United States). The samples were then examined by CyTOF2 mass cytometry (Fluidigm, United States).