The suspension of fixed, permeabilized, barcoded cells was stained using either anti-human CD3 (UCHT1) phycoerythrin (PE)–cyanine 7 (Cy7) (0.5 μl/ml; eBioscience; KP and TI) or anti-human CD3 (UCHT1) allophycocyanin (APC) (1 μl/ml; eBioscience; DR and CV) and distributed across a 96-well polypropylene plate. The cells were stained using a Biomek NX liquid handler (Beckman Coulter) with fluorescently conjugated anti-human antibodies against intracellular signaling epitopes (table S1) for 45 min in the dark at room temperature, as per the manufacturer’s instructions. Antibodies were purchased from BD Biosciences, Cell Signaling Technology, Merck Millipore, and Bioss. Antibodies against intracellular epitopes were used either individually (KP, DR, and CV) or in groups of three antibodies per plex (TI). The cells were washed twice and resuspended in FACS buffer at 1 × 106 cells/ml for acquisition.

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