Rhodium dna intercalator

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.

Antibodies not purchased from Fluidigm were labeled using metal tags using Maxpar Antibody Labeling Kits (Fluidigm). Antibody titration was undertaken and used at a concentration from 0.25 to 0.5 μg/mL. One to three million cells were first stained per sample with a solution containing rhodium DNA intercalator (Fluidigm) to distinguish live/dead cells before Fc receptor blocking (Miltenyi Biotec). Samples were then stained with the mixture of conjugated antibodies for cell-surface antigens. After washing in Maxpar Cell Staining Buffer (Fluidigm), samples were fixed and permeabilized using the Foxp3 Transcription Factor Staining Buffer Kit (Thermo Fisher) prior to washing and incubation with metal-conjugated antibodies recognizing intracellular antigens. Samples were washed twice in cell-staining buffer, fixed by incubation with 1.6% paraformaldehyde (PFA) (Pierce) for 10 min, and finally incubated overnight with iridium DNA intercalator in Maxpar Fix and Perm Buffer (Fluidigm). Samples were washed again, being acquired on a Helios mass cytometer (Fluidigm). After acquisition, .fcs files were normalized using tools within Helios software, and gating and downstream analysis using clustering and dimensionality reduction algorithm viSNE was performed using Cytobank (https://www.cytobank.org/).