PBMC (1 × 106/mL) are cultured in RPMI 1640 supplemented with 10% human serum, 2 mM L-glutamine, and 1% penicillin (Invitrogen Ltd, Paisley, UK) and incubated at 37°C in a humidified 5% CO2 atmosphere for 2 h in a 12-well plate. After 2 h, non-adhering PBMCs are harvested and discarded; monocytes (adhering cells) are culture in medium alone (unstimulated) or primed with 2 μg/mL LPS for 2 h (Sigma–Aldrich, St. Louis, MO) before stimulation with Nigericine (5 μM) (Sigma–Aldrich) for 1 h at 37°C in a humidified 5% CO2 atmosphere. Adhering cells (monocytes) are then collected by trypsin treatment and prepared for FlowSight analysis by immunofluorescence staining as THP1-derived macrophage (see above).