PBMC proliferation assay

PBMCs were isolated as described above, and plated into Falcon 96-well tissue culture plates (Corning, Corning, NY). PBMCs were plated in triplicate at a final density of 2.5 × 10⁵ cells/well in a 200 µL final volume for four conditions: (1) with media only (i.e., unstimulated proliferation), (2) with 1 µg/mL of lipopolysaccharide (LPS) obtained from E. coli (serotype 026:B6, Sigma-Aldrich, St. Louis, MO), (3) with 5 µg/mL of phytohemagglutinin (PHA; Sigma-Aldrich, St. Louis, MO), and (4) with 50 µg/mL of polyinosinic:polycytidylic acid (poly (I:C); high molecular weight; InvivoGen, San Diego, CA). Plates were incubated at 37 °C, 5% CO₂, and 100% humidity.

Approximate cell density was measured at three time-points: 24, 48, and 72 h post-plating using the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega, Madison, WI). Each plate was read on a plate reader (BMG LabTech FLUOstar Omega, Cary, NC) at 490 nm.