PBMC and monocyte isolation.

A quantity of 30–60 mL fasting blood was collected into EDTA-containing Vacutainers (Becton Dickinson; catalog 364606). Blood then was diluted (1:1) with RPMI medium (Corning; catalog 17-105-CV) and applied to Histopaque gradient medium (MilliporeSigma; catalog 10771) using SepMate-50 (STEMCELL Technologies), and centrifuged at 1200g for 10 minutes. The top layer contained the enriched PBMCs, which were collected followed by centrifugation at 300g for 10 minutes. The pellet was resuspended with ACK lysis buffer (Gibco; catalog A1049201) and incubated at room temperature for 5 minutes to remove residual red blood cells. Next, the enriched PBMCs were washed twice with RPMI medium. All PBMC samples were subjected to the baseline Seahorse mito stress test. When available, the remaining cells were used for cytokine mRNA quantitative PCR and in vitro assays. For monocyte isolation, a magnetic bead–based negative-selection monocyte isolation kit (Miltenyi Biotec, 130-096-537) was used per manufacturer’s instructions. Briefly, antibody-conjugated magnetic bead solution was added to isolated PBMCs resuspended in RPMI containing 0.5% BSA and incubated for 20 minutes at 4°C, followed by magnetic column binding and elution. The procedure was repeated once to improve the purity of monocytes. The monocytes were washed once with RPMI before subsequent experiments.