Changes in mitochondrial membrane potential (ΔΨm) after EHV-1 infection. Monocyte-enriched fractions of equine PBMC (5 × 105 cells) were infected for 4 (left panel) or 24 h (right panel) with the RacL11 strain of EHV-1 at an MOI of 1 (A) or 5 (B). Positive apoptosis and stimulation controls were sodium azide (25 mM) and LPS (10 ng/mL), respectively (C, only one time point for each control shown), whereas mock-infected cells served as a negative control (green overlay). Changes in ΔΨm were evaluated with DePsipherTM and flow cytometry. With this technique, decreased red fluorescence (FL2) indicates altered ΔΨm, an early marker of apoptosis. Data shown is representative of 3 different experiments. Marked changes in ΔΨm, indicative of apoptosis, were evident in cells infected with RacL11 at an MOI of 5 after 24 h and sodium azide controls, but not in cells infected with RacL11 for 4 h at either MOI or 24 h after EHV-1 infection with an MOI of 1 or treatment of cells with LPS.