OA cells were cultured to confluence in 10-cm dishes. On the collection day, cells were stained with 25 μM Idu (5-Iodo-2′-deoxyuridine) for 15 min at 37°C in the cell incubator and then with 0.5 μM cisplatin for 5 min at RT. Cells were then lifted with 0.25% trypsin-EDTA (Gibco) for 15 min at 37°C. Trypsin was quenched using media containing 10% FBS, and cell were washed three times with phosphate-buffered saline to remove any trace amounts of trypsin. Cells were fixed after straining through a 35 μM strainer in 1.6% paraformaldehyde (PFA) for 10 min at RT. Cells were washed four times with cells staining media, counted, and frozen in 1 million–cell aliquots in a small amount of cell staining media at −80°C. To stain, cells were thawed on ice and barcoded using the Cell-ID 20-plex Pd Barcoding Kit (Fluidigm) according to the manufacturer’s specifications. After barcoding, cells were labeled as previously described (8). Briefly, all barcoded samples were combined into one FACS tube and washed 3 times with cell staining media and stained with the cell surface antibodies for 30 min at RT according to the concentrations in table S1. Cells were then washed 2 times with cell staining media and permeabilized with 1 ml of cold methanol added dropwise with continuous gentle vortexing. Cells were incubated for 10 min on ice, with gentle vortexing every 2 to 3 min to avoid cell clumping, then washed in cell staining media, and stained with the intracellular antibodies for 30 min at RT. After 2 times washed with cell staining media, cells were resuspended in 1.6% PFA with Cell-ID Intercalator-Ir (Fluidigm) used at 1:2000. Cells were measured using the cyTOF 2 (Fluidigm) and injected using the supersampler. EQ (Four Element Calibration) beads (Fluidigm) were added just before runtime (1:10 dilution) to normalize signal over runtime.

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