Maxpar labeling kit

Antibody panel design (including the description of antibodies, concentration, clone information and metal isotype tag used) are provided in Supplementary Table 1. All antibody–metal conjugations were conducted with the Maxpar labeling kit (Fluidigm). Antibody concentration was titrated (100–500 µg ml⁻¹) using a Nanodrop (Thermo Scientific), and conjugated antibodies were stored in a Candor antibody stabilizer (Candor Bioscience) at 4 °C (ref. ^{11 (link)}). Antibody staining patterns and concentration were evaluated by inspection of IMC images from a variety of tissues, including tonsil, normal breast and breast cancer.

Carrier-free antibodies were conjugated to metal tags using the MaxPar^® labeling kit (Fluidigm) following the manufacturer’s instructions. Antibodies were stored at 500 μg/ml in a stabilizing solution (Candor Biosciences) at 4°C.

The antibody panel included those related to lymphocyte types, cytokine expression, lymphocyte activation, and vascular and spatial structures of cells from other tissues. Descriptions of the antibodies, isotope tags, clones, and concentrations used for staining are provided in Supplementary Table 2. Metal-conjugated primary antibodies were applied using a Maxpar labeling kit (Fluidigm, USA). Concentration was determined using a Biotek instrument (Berten Instruments) and was adjusted to 0.5 μg/μL in Antibody Stabilization Solution (Candor Bioscience GmbH, Wangen, Germany). Antibodies were stored at 4°C for long-term storage.

CyTOF staining was conducted using single‐cell suspensions derived from bladder tumor specimens (n = 21 patients with muscle‐invasive [≥T2] urothelial carcinoma of the bladder, Table 1) according to manufacturer's instructions. To define the phenotypic diversity of human bladder tumor innate lymphoid cells, we designed a CyTOF panel of 36 antibodies (Table S1).⁸ As described,⁸ cells were thawed in Hank's balanced salt solution without Ca2⁺ or Mg2⁺ + 10% FBS and the number of viable cells was quantified using trypan blue. Before surface staining, cells were stained with cisplatin or discrimination of dead cells from live cells. Cells were then stained first with the surface antibody cocktail for 30 min (see Table S1 for clone list and metal). After washing, cells were fixed and permeabilized with MaxPerm‐S buffer for 30 min before staining with the intracellular antibody cocktail for 30 min. After washing steps, cells were stained for Cell‐ID Intercalator‐Ir to discriminate single nucleated cells from doublets. Finally, cells were resuspended in Cell Acquisition Solution (CAS)‐bead solution to 1 million cells/mL before the acquisition of data on Helios. Purified antibodies lacking carrier proteins were conjugated using the Maxpar labeling kit and according to the protocol provided by Fluidigm.