Pt 198

60‐well barcoding scheme used for 4 cell line dataset: prepared according to (Zunder et al, 2015): 8 choose 4 barcoding scheme with following metals and stock concentrations: 102Pd (10 µM), 104Pd (15 µM), 105Pd (20 µM),106Pd (20 µM), 108Pd (20 µM), 110Pd (15 µM), 113In (20 µM), and 115In (20 µM) in DMSO (Sigma).

126‐well barcoding scheme used for overexpression dataset: prepared according to (Zunder et al, 2015): 9 choose 4 barcoding scheme with following metals and stock concentrations: 89Y (10 µM), 103Rh (200 mM), 105Pd (10 µM), 106Pd (10 µM), 108Pd (10 µM), 110Pd (10 µM), 113In (20 µM), 115In (10 µM), and 209Bi (2 µM) in DMSO.

To extend the barcoding capacity, spheres from multiple plates are collected and either barcoded with different monoisotopic cisplatin (Pt 198, Pt194, Fluidigm).

Spleens were harvested from mice and the tissue was homogenized. Cells were washed twice with PBS and counted with the ViCell XR analyzer and resuspended in wash buffer (0.5% BSA in PBS). A cell surface antibody mix was added to each sample and put on the shaker for 1 h at room temperature. Cells were then washed three times to remove residual antibodies and 1 μM Pt198 (Fluidigm), a live/dead stain, was added to each sample for 5 min on the shaker at room temperature. Cells were washed three times, then 4% paraformaldehyde was added to each sample and were incubated for 15 min at room temperature. Cells were washed once, then 1 mL 100% methanol was added to each sample overnight. After one wash, intracellular antibody mix was added to each sample with incubation for 1 h on the shaker at room temperature. Cells were washed two times, then Ir‐intercalator‐193, 125 μM (Fluidigm, California, USA) was added to each sample with a final dilution of 1:2000. Samples were analyzed with the Fluidigm Helios CYTOF Mass Cytometer.