Incucyte s3 live cell analysis system

Cell growth was determined by the measure of cell confluence over time. Four pictures/well were taken by the Incucyte S3 Live-Cell Analysis System (Essen Bioscience) every 6 h, and data were analyzed using the Incucyte S3 Live-Cell Analysis System looking at the cell confluence per image.

HCC827 cells from POT, DMSO7, DMSO8, GEF1–3 and TRM4–6 were seeded at a density 1000 and 3000 per well in a 96-well and 48-well plates (Corning), respectively. Cells were grown in six independent replicates in 96-well plate and three independent replicates 48-well plates for the duration of total 8 days. Media containing neither a vehicle control nor a drug were used as a fresh media every 3 days. The plates were placed into the IncuCyte® S3 Live-Cell Analysis System (Sartorius) and images were taken every 2 h at ×4 and ×10 magnifications. At the end of 8 days, confluency determination for each of the time points were automatically calculated based on the images acquired using IncuCyte® S3 Live Cell Analysis System (Sartorius). We calculated the growth rate of each line using linear fitting of log-transformed data (see Supplementary Fig. 15).

NT, ΔCTSL, and ΔWDR81 cells were grown in 6-well plates (Greiner Bio-One). The cells were adsorbed with VSV (GFP) or VSV-EBO GP (GFP) at 5 PFUs/cell for 1 h at room temperature. After 1 h, the cells were washed three times with PBS and incubated in growth medium at 37°C in an IncuCyte S3 Live-Cell Analysis System (Sartorius). At the indicated times post infection, three images per well were acquired using a X10 objective and green (excitation [440–480 nm], emission [504–544 nm]) and phase channels. The images were analyzed using the IncuCyte S3 Live-Cell Analysis System (Sartorius) software and plotted as GFP positive cells (per image) / Percent cell confluency versus Time post infection (h).

For proliferation and cytotoxicity assays, the cells were seeded at a density of 6.25 × 10³ cells/cm² and 1.56 × 10⁴ cells/cm², respectively, and allowed to adhere for 24 h before the addition of the conditions described before, based on the guidelines of the IncuCyte^® S3 Live-Cell Analysis System (Sartorius) and comparable to other studies [17 (link),20 (link)]. After 24, 48, and 72 h exposure to the different conditions, proliferation and cytotoxicity were studied using the IncuCyte^® S3 Live-Cell Analysis System (Sartorius) according to the manufacturer’s guidelines and other studies [22 (link)]. For the cytotoxicity experiments, the Incucyte^® Cytotox Green Reagent diluted in standard medium (1:40,000, Sartorius) was used. Images were taken every two hours for three days with a 10× lens and each condition was run in triplicate. For proliferation and cytotoxicity experiments, phase images were acquired using phase contrast. For cytotoxicity experiments, fluorescent images were acquired using a green fluorescence channel with 400 ms exposure. Proliferation was expressed as % confluence. Cytotoxicity was calculated as the total Cytotox Green area (µm²) divided by % confluence.