Cell id intercalator ir

Tissue processing and antibody staining was performed as described in detail in (21 (link)). In short, 5-μm cryosections of fresh frozen lungs were fixed (Image-iT Fixative Solution, Thermo Fisher Scientific) and stained with the antibody panel listed in Table 3 and Cell-ID Intercalator-Ir (Fluidigm). Scanning of the (dried) slides was done with the Hyperion Imaging Mass Cytometer (Fluidigm). Images are available at the Figshare repository https://doi.org/10.25418/crick.19590259.
Image processing was performed with the previously described 1-pixel expansion single-cell segmentation pipeline using imcyto (nf-core/imcyto). The resulting single-cell data were clustered with Phenograph and subsequently annotated to the different cell types (table S1).

The panel of monoclonal antibodies used in this study is described in detail in Supplementary Table I. When indicated, purified carrier-free antibodies were conjugated using Maxpar^® antibody labelling kit (Fluidigm) according to the manufacturer’s instructions.
PBMCs (4x10⁶) were washed in PBS and stained with 2.5 µM Cell-ID™ Cisplatin (Fluidigm) for 5 min at room temperature to identify dead cells. Cells were then washed in Maxpar^® Cell staining buffer (CSB, Fluidigm) and incubated in an extracellular staining cocktail for 30 min at 4°C. PBMC were washed in CSB and resuspended in FOXP3 Fixation/Permeabilization buffer (Thermofisher Scientific) for 40 min. Cells were then washed with Permeabilization buffer (Thermofisher Scientific) and stained with an intracellular staining cocktail for 30 min at 4°C. Samples were then washed in Permeabilization buffer. Finally, cells were stained with 125nM Ir191/193 DNA intercalator (Cell-ID Intercalator-Ir, Fluidigm) and acquired with a CyTOF2^® instrument. Data were normalized using EQ Four Element Calibration Beads (Fludigm).

These samples were processed and stained as described in Amir et al.^{68 (link)}. Briefly, frozen aliquots of cells were thawed in 98% IMDM + 2% FBS + DNase I. Thawed cell pellets were immediately re-suspended in PBS containing 5 µM Cell-ID Cisplatin (Fluidigm) and incubated for 5 min at room temperature. Cells were washed one time with cell staining buffer and fixed using 2% formaldehyde for 10 min at room temperature. Fixed cells were washed with cell staining media (low barium PBS + 0.5% bovine serum albumin + 0.02% sodium azide) and incubated for 30 min at room temperature in surface epitope metal conjugated antibodies (Supplementary Table 4). Surface stained cells were washed twice and re-suspended in cold methanol for 30 min at 4 °C. Methanol permeabilized cells were washed one time and re-suspended in intracellular epitope metal conjugated antibody mix for 1 h at room temperature. Stained cells were washed once before incubation in 0.5 µM Cell-ID Intercalator-Ir (Fluidigm). Fixed and stained cells were washed two times in cell staining media and re-suspended at (2.5–5) × 10⁵ /ml in water before being run on CyTOF instrument.
Data was analyzed utilizing the SPADE3 algorithm which was used to visualize and interpret data^{69 (link)}. The analysis was carried out in MATLAB per the original references. The Auto Suggest Annotation of SPADE3 was utilized to decide clustering.

Single-cell suspensions derived from freshly isolated tumors were prepared by mechanical dissociation with a gentleMACS Dissociator (Miltenyi Biotech) and enzymatic digestion with collagenase II and dispase II. Cells were incubated with 25 μM cisplatin, followed by staining at room temperature for 30 min with heavy metal-conjugated antibodies provided by CyTOF core at the Penn Institute for Immunology. Cells were fixed with 1.6% paraformaldehyde and stained with Cell-ID Intercalator-Ir (Fluidigm) and analyzed using a CyTOF mass cytometer (Fluidigm), followed by analysis with Cytobank software (7.3.0).