Live-dead assay on EHV-1-infected or control-treated monocyte-enriched fractions of peripheral blood. Representative flow cytometric analysis of Annexin V and propidium iodide staining of monocyte-enriched fractions of equine PBMC (5 × 105 cells) infected for 24 h with RacL11 (MOI of 5, right panel). Vehicle (PBS, left panel) and LPS (10 μg/mL, middle panel) served as negative and positive stimulation controls, respectively. Monocytes were identified and gated (R1) by their characteristic FSC and SSC. A second population of cells with lower FSC and similar SSC was identified and similarly gated (R2; upper panel). The percentage of dead cells in each gate was determined by combining the total percentage of cells positive for Annexin V (lower right quadrant), propidium iodide (upper left quadrant) or both Annexin V and propidium iodide (upper right quadrant) in fluorescence dotplots (lower two panels). In this representative experiment (of n = 4), the degree of cell death in the monocyte gate was similar for EHV-1-infected (15%), PBS- (19%) or LPS-treated (13%) cells. The R2 gate consisted mostly of dead cells (EHV-1: 98%; PBS: 98%, LPS: 94%).