Glass coverslip dishes

RBL2H3 were plated on glass coverslip dishes (MatTek, Ashland, MA) and incubated with 1μM Fluo-4 for 30 minutes at 37°C in a standard modified Ringer's solution as described above. After washing, cells were stimulated as indicated on a 37° C heated stage. Calcium signals were acquired using a Nikon Ti Eclipse confocal microscopy system, using EZ C1 software for acquisition and NIS Elements software (Nikon) for analysis. Where indicated, nominally calcium-free external conditions were achieved by the preparation of 0mM added CaCl₂ Ringer solution containing 1mM EGTA.

Live imaging was performed as previously described on a Zeiss AxioObserver.Z1 widefield epifluorescence microscope equipped with a heated environmental chamber, 40×/1.3NA objective, and DAPI/FITC/Texas Red/Cy5 filters (Spence et al., 2016 (link)). Briefly, purified DiD-labeled VSV expressing full-length EBOV GP was incubated with 100 μg/mL CA45 in imaging buffer for 1 hour at 37°C. Virus was spinoculated at 4°C and 1500 × g for 20 minutes onto pre-chilled U2OS cell monolayers seeded on glass coverslip dishes (MatTek). Following the removal of unbound virus, dishes were immediately mounted in the microscope, and warm imaging buffer (140 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 20 mM HEPES, 5 mM glucose, 2 μg/mL Hoechst 33342, and 2% FBS, pH 7.4) was added to mark the start of experiments. Images were taken every 6 seconds over a two-hour period. To examine viral trafficking, antibody-treated DiD-labeled virus was spinoculated onto U2OS cells stably expressing monomeric NeonGreen-tagged NPC1. Cells were imaged live at 45 minutes after warming to 37°C. Data were compiled from three independent experiments, and co-localization analysis was performed using Volocity (Perkin Elmer) software as described previously (Spence et al., 2016 (link)).