WT mouse neutrophils were preincubated with (A) DPI, (B) ML-7, or DMSO buffer as a control and cocultured with ΔgD-2 or control immune serum pretreated HSV-infected, calcein-labeled Vero target cells. The percent cytotoxicity was quantified by measuring cell-free calcein in ΔgD-2 minus control serum treated targets (n = 6–8). (C) HSV-infected Vero cells were labeled with Vybrant DiI dye, incubated with ΔgD-2 or control immune serum, and neutrophils isolated from WT, Hvem^–/–, or Light^–/– mice. The percentage of DiI uptake was measured by flow cytometry on live neutrophils incubated with ΔgD-2 versus control immune serum (n = 6). (D) DiI-labeled (green) and calcein-AM–labeled (red) HSV-infected Vero cells were incubated with ΔgD-2 immune serum with neutrophils isolated from WT, Hvem^–/–, or FcγRIV^–/– neutrophils. Cytotoxicity was imaged over a 2.5-hour incubation with the Operetta CLS imaging system with images collected at 20 × magnification over 3 fields (n = 3). Representative images are shown at 3 timepoints: 22.5, 28.5, and 120 minutes after the start of imaging. (E) Fluorescent intensity of DiI⁺ neutrophils was measured with the Spot Intensity Analysis plugin using ImageJ across 3 fields (n = 3; orange, blue, and green) for WT, Hvem^–/–, and FcγRIV^–/–. Fluorescent intensity is reported for individual DiI⁺ neutrophils and the mean intensity in each field. Cytotoxicity and %DiI⁺ neutrophil were compared by repeated measures or 1-way ANOVA, respectively with Holm-Šidák’s multiple comparisons test; Fluorescent Intensity was compared by 1-way ANOVA with Holm-Šidák’s multiple comparisons test against the means of each field (**P < 0.01, ***P < 0.001, ****P < 0.0001).