Normalization beads

To avoid batch variation effects, all samples were stained with the same batch of antibodies and on the same day. All samples were acquired in 1 day to avoid instrument signal fluctuation. Antibodies (listed in Table S1) were either pre-conjugated from the manufacturer (Fluidigm, San Francisco, CA) or conjugated in-house with the appropriate metal isotopes as previously described (24 (link)). Cells were thawed and 5.10⁶ PBMCs were transferred per well. As previously described (24 (link)), PBMCs were incubated with Rhodium DNA-intercalator (Fluidigm), first stained with the primary surface antibody mix for 1 h, and then stained with the secondary surface antibody mix for 15 min. Next, samples were resuspended in 1.6% PFA and incubated 20 min. Cells were finally incubated 30 min in permeabilization buffer with 1 μM Iridium DNA-intercalator before a 4°C overnight incubation in 1.6% PFA with 0.1 μM Iridium DNA intercalator. For acquisition, cells were washed and filtered through a cell strainer cap of a 5-ml polystyrene round-bottom tube (BD Biosciences). Normalization beads (Fluidigm) were added to each sample. Then, samples were acquired using a mass cytometer (CyTOF-I; Fluidigm) and following the standard procedure recommended by the manufacturer. An average of 200,264 ± 13,472 events was acquired per sample.

Cryopreserved PBMCs were thawed, rapidly transferred to warm X-VIVO™ containing a nuclease (Benzonase® Nuclease, Merck Millipore, 25 U/mL), followed by centrifugation and resting in X-VIVO™ for 4 h at 37°C, 5% CO₂. The resting time was optimized in set-up experiments (data not shown). Viability staining was performed according to the manufacturer's instructions with Cell-ID™ cisplatin (Fluidigm). PBMCs from each individual were split into two aliquots; one was not stimulated, and the other was stimulated with 50 ng/mL TNF for 12 min. Stimulation time and dose had been defined after a series of TNF time and dose titrations. Cells in both samples were fixed in proteomic stabilizer (Smart Tubes Inc.) for 10 min and stored at −80°C until barcoding and staining. All cells were barcoded simultaneously with 20-plex Cell-ID™ barcoding kits (Fluidigm) as recommended by the manufacturer. After pooling, surface staining, methanol-permeabilization, and intracellular staining were carried out. PBMCs were then stained with MaxPar DNA intercalator overnight (Fluidigm) and analyzed the following day on a Helios mass cytometer (Fluidigm) after addition of normalization beads (Fluidigm). Raw FCS-files were bead-normalized, concatenated, and debarcoded with software tools from Fluidigm before subsequent analysis.