Glass chamber slide

Cells were plated on 2-well or 4-well glass chamber slides (Thermo Fisher) coated with fibronectin (100 μg/mL in PBS, Millipore). Cells were maintained in complete media at 37°C and 5% CO₂ in an environmentally controlled chamber. After PM damage, cells were fixed cells in 4% paraformaldehyde-PBS 10~20 min at room temperature, permeabilized with 0.3% Triton X-100 in 1X PBS, blocked in 3% BSA in 1X PBS, stained with primary antibodies for 2–4 hr (room temperature) to overnight at 4°C, followed by staining with 2° antibodies (1 hr) and DAPI stain. Samples were imaged using Lionheart microscope (Biotek).

Virus-mediated cell membrane FFWI was performed as previously described (Guirakhoo et al., 1993 (link)). Essentially, C6/36 cells were seeded onto glass chamber slides (ThermoFisher, Waltham, MA) before infection with VEEV TC83 at an MOI of 1.0 and incubation at 28 °C/5%CO₂ while maintaining the pH at 7.5. Twenty-four hours after infection, cells were incubated with various concentrations of MAb diluted in BA-1 for 2 h at 28 °C/5%CO₂. After MAb treatment, cells were exposed to fusion medium (DMEM buffered at pH < 5.0 with 2-N-morpholinoethanesulfonic acid) for 1 h, after which fusion medium was replaced with DMEM at neutral pH. Cells were incubated another 24 h at 28 °C/5%CO₂ before being fixed in absolute methanol and stained using the Wright-Giemsa stain kit (Abcam, Cambridge, MA). An isotype MAb (100 μg/ml), virus-infected non-treated cells at pH 7.5, and uninfected MAb-treated cells at pH 5.0 were included as controls. The number of nuclei and the number of cells in five microscopic fields (magnification 100-fold) were counted and the fusion index [1 - (number of cells/number of nuclei)] was calculated. A one-way ANOVA with Tukey’s multiple comparisons test was used to compare differences in percent fusion.