Intercalator ir

All antibodies were obtained from Fluidigm Maxpar Human Peripheral Blood Basic I Phenotyping Panel Kit (Catalog #: 201302). For cell staining protocols, Cell Staining Buffer (CSB) (Catalog #: 201068), Intercalator-Ir (Catalog #: 201192A, 125 μM), water (Catalog #: 201069), and Cell Acquisition Solution (CAS) (Catalog # 201240) were all acquired from Fluidigm (San Francisco, Ca). Pierce^™ 16% formaldehyde (Catalog #: 28908) was purchased from Thermo Fisher Scientific (Waltham MA) to make a working concentration of 1.6% in PBS. Reagents for sample analysis on the Helios mass cytometer included Tuning Solution (Catalog #: 201072), EQ^™ Four Element Calibration Beads (EQ4, Catalog #: 201078), and Washing Solution (Catalog #: 201070) were also acquired from Fluidigm. Five Antibody Binding Capacity (ABC) calibration beads were synthesized using dispersion polymerization, independently characterized for the number of metal atoms per bead [44 (link)] and combined for use in mass cytometry. The five beads feature a roughly equivalent amount of Ce, with zero and then logarithmically increasing amounts of La, Eu, Ho, and Lu.

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).

Descriptions of cell markers and isotope tags are provided in Supplementary Tables 2–5. Staining was performed according to Fluidigm IMC recommendations for FFPE as follows. Briefly, tissue sections were dewaxed in xylene and rehydrated in a graded series of alcohol. Epitope retrieval was conducted in a water bath at 96 °C in Tris-EDTA buffer at pH 9 for 30 minutes, then cooled and washed in metal-free PBS. Blocking with Superblock (ThermoFisher) plus 5% FcX TruBlock (Biolegend) was followed by staining with antibody cocktail prepared in 0.5% BSA and metal-free PBS overnight at 4 °C. Sections were washed in 0.02% TritonX100 followed by metal-free PBS, then nuclear staining was performed using 1:200 or 1:300 dilution of Intercalator-Ir (125 μm, Fluidigm) solution for 30 min, followed by ddH₂O for 5 min. Slides were air-dried before IMC measurement.
The abundance of bound antibody was quantified using the Hyperion imaging system (Fluidigm) controlled by CyTOF Software (version 7.0.8493), with UV-laser set at 200 Hz. Count data were then converted to tiff image stacks for further analysis using MCD Viewer (version 1.0.560.6, Fluidigm) or imctools (Bodenmiller lab, https://github.com/BodenmillerGroup/imctools).

Combined samples were washed once with CSB and incubated with Human Fc Receptor Binding Inhibitor Antibody (Thermo Fisher, Waltham, MA, USA) for 10 min at RT to lower non‐specific binding. Anti‐human ICOSL‐biotin (BioLegend, San Diego, CA, USA) was added to the samples for incubation for another 30 min at RT. These cells were washed twice with CSB and stained with 29 metal isotope‐tagged antibodies and 1 metal‐labelled antibody against biotin (Supplementary table 2) for 30 min at RT. These stained cells were washed thrice with CSB and incubated with 1 mL Fix & Perm Buffer (Fluidigm) containing 125 nm Intercalator‐Ir (Fluidigm) overnight at 4°C.

Tissue samples were formalin-fixed and paraffin-embedded at the University Hospital of Basel. Sections were baked for 1 h at 60°C, dewaxed in fresh xylene for 20 min, and rehydrated in a graded series of alcohol (100%, 95%, 80%, 70%; 5 min each). Antigen retrieval was carried out in IHC Antigen Retrieval Solution pH 9 (Invitrogen) for 30 min in a 95°C water bath. Sections were cooled and then immediately blocked with 3% BSA for 45 min at room temperature. Samples were stained for 5 h at room temperature using the Maxpar Human Immune Activation IMC Panel Kit (Fluidigm, South San Francisco, CA) in combination with the Maxpar Human Tumor-Infiltrating Lymphocytes IMC Panel Kit (Fluidigm) and metal-conjugated anti-Vimentin (¹⁴³Nd, clone D21H3, Fluidigm). Tissue sections were washed twice with 0.05% Tween-20 in Maxpar PBS (Fluidigm) before staining with Intercalator-Ir (Fluidigm) for 30 min at room temperature. Slides were then washed with Maxpar water for 5 min and let air-dry for at least 20 min at room temperature before acquisition.