In cell 2200 automated

The cells were cultured and treated as described above. On the day of the experiment, cells were live-stained with 100 μl/well of 50 nM LysoTracker Red DND-99 dye (Invitrogen) in the medium at 37 °C for 1 h, followed by fixation in 100 μl/well 4% paraformaldehyde (Sigma) for 15 min. and twice washes with PBS. The nuclear staining was performed by an addition of 100 μl/well of 1 μg/ml Hoechst 33342 (Invitrogen) in PBS and incubation at room temperature for 15 min. The images were taken by the IN Cell 2200 automated fluorescence plate imaging reader (GE Healthcare) with ×20 or ×40 objective lens, and imaging detection was performed using DAPI (nucleus) and TRITC (LysoTracker) filter sets. Montages were generated using Fiji-ImageJ (NIH).

DePsipher Mitochondrial Potential Kit (6300-100-K, R&D system) was applied to monitor the mitochondrial potential after ZIKV infection. Cells were plated in black 96-well plates with clear bottom and infected with ZIKV at MOI = 0.5, 1, or 2. On the day of the experiment, the DePsipher^TM solution was prepared according to the manufacturer’s instructions. Before DePsipher staining, the nuclear staining was performed by the addition of 100 μl/well of 1 μg/ml Hoechst 33342 (Invitrogen) in medium and incubation at 37°C for 15 min. After that, the medium was removed, and cells were covered with diluted DePsipher^TM solution, followed by incubating at 37°C for 15 min. Then, cells were washed with 100 μl of prewarmed 1× reaction buffer with Stabilizer Solution. The images were taken by the IN Cell 2200 automated fluorescence plate imaging reader (GE Healthcare) with 20× or 40× objective lens, and imaging detection was performed using DAPI (nucleus), FITC (DePsipher monomer) and TRITC (DePsipher aggregates) filter sets. Montages were generated using Fiji-ImageJ (NIH).