Activation and Labeling of PBMCs

PBMCs were rested in 37°C 5% CO₂ incubator for 30 min (biosynthesis activity assays), 1 h (metabolism assays), or 2 h (stimulation assays) at 5 × 10⁶ cells/mL in CCM. Metabolism samples were fixed in 1.6% PFA in PBS for 10 min and then palladium barcoded as previously described (Zunder et al., 2015 (link)). After rest, biosynthesis activity assay samples were spiked with 2 mM BRU and 10 μg/mL puromycin and incubated for an additional 30 min (Kimmey et al. 2019 (link)) and then fixed and palladium barcoded. Stimulation samples were resuspended in CCM with 3.3 mM H₂O₂ (Irish et al., 2010 (link)) and specified doses of anti-kappa F(ab’)₂ (Southern Biotech) and CD40L (BioLegend) for 10 min and then fixed and palladium barcoded.