Stimulation is defined broadly as the exposure of PBMC cells to a ligand that has the potential to perturb resting-state cell signaling dynamics by either increasing or decreasing the expression of cell signaling epitopes. PBMCs were pelleted and resuspended [via 30-μm cell strainer (Partec) for KP and TI studies] in 96-well polypropylene plates using complete RPMI media without penicillin-streptomycin at 2 × 106 cells/ml (KP), 0.75 × 106 cells/ml (TI), and 0.4 × 106 cells/ml (DR and CV). The cells were rested for 75 min (KP and TI) or 45 min (DR and CV) at 37°C before ligand exposure. For the DR and CV studies, this resting period was used to preincubate the cells with the extended FDA-approved library compounds and screening hits, respectively, before stimulation. A standardized sample amount of 5.25 × 106 PBMCs per donor was used in the TI study, and no significant differences were observed in T cell frequencies between the control (67.1 ± 12.9%), pretreatment (72.0 ± 11.9%), and posttreatment (74.0 ± 9.8%) SCZ groups (P > 0.27; Wilcoxon rank sum and signed-rank tests).

Stimulants and vehicle were reconstituted in complete RPMI media without penicillin-streptomycin and added to the cells using a Biomek NX liquid handler (Beckman Coulter) with an integrated compact shaker-heater-cooler system (Inheco). The final concentrations of DMSO in all conditions including vehicle were 0.1% (KP and TI) and 0.2% (DR and CV). Vehicle wells represented one-eighth of the total wells assayed (KP and TI), one-sixth of the total wells assayed (DR), or one-quarter of the total wells assayed (CV) and were spaced evenly across each 96-well plate. The cells were exposed to the stimulants at 37°C for 1, 5, 15, and 30 min (KP) or 30 min (TI, DR, and CV). Ligand exposure was halted by fixation for 10 min at 37°C using paraformaldehyde (Sigma-Aldrich) in PBS at a final concentration of 1.6%.

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