Spark cyto 400

Statistical analyses were performed using GraphPad Prism 9 (GraphPad Software, San Diego, CA USA). Results were considered statistically significant when p < 0.05.
For infection kinetics, mean values of confluence measures of BAC and respective CI measured using Spark Cyto 400 multimode plate reader (Tecan) were compared individually for each time point by multiple two-way t-tests (Table S6). TCID₅₀ values were compared using one-way analysis of variance (ANOVA) We furthermore compared vRNA levels in A549-AT cells at different time points using Welch t-test (Table S2). NT₅₀ results of the recombinant variant and respective clinical isolate obtained by microscopical analysis were compared through one-way (ANOVA). Dose-responses for monoclonal antibodies and antiviral compounds non-linear regression method was used. Regression was performed on 6 and 3 independent data points for mAb treatment and inhibitor treatment, respectively. Derived IC₅₀ values are depicted in the respective graph. Using the same software, unpaired t-tests were performed for statistical analysis. Multiple comparisons were corrected by using the incorporated Bonferroni–Dunn method. For analyses with HBEpCs one-way ANOVA was used to determine statistically significant differences.

CaCo2 and A549-based cells were seeded in 12-well plates (2 × 10⁴ A549-AT and 1 × 10⁵ Caco2/well, respectively). Cells were incubated for 16 days at 37°C and 5% CO₂ and cell count and confluency was optically determined using SparkCyto 400 (Tecan) at regular intervals starting after 48 h after seeding. To allow attachment, cells were incubated for 48 h before monitoring. Prior to imaging the wells were washed using PBS and refilled with 2 ml culture medium. Trypsinization assay was performed in 96 well plates. After rinsing twice with PBS, confluent cells were treated with 100 μl trypsin (2 mg/ml) and incubated for different intervals. Cells were incubated at room temperature and 37°C for incubation times longer than 10 min. Subsequently cells were rinsed with cell culture medium, remaining cells were fixed with 3% PFA, and nuclei were stained with DAPI and analyzed with a plate reader (SparkCyto 400).

Hoechst 33342 (10 μg/mL final concentration) and/or Mitotracker Orange (100 nM, ThermoFisher) were added to cells in growth medium and plates incubated in the dark (37 °C, 5% CO₂). Following a 30-min incubation, the medium was aspirated, the cells washed in sterile PBS, and the fresh growth medium returned. Stained cells were imaged using an EVOS M5000 microscope and counted using the SparkCyto-400 plate reader (TECAN, Männedorf, Switzerland). Mitotracker Orange staining was further visualized using the Leica DM4000 upright fluorescence microscope (Leica, Wetzlar, Germany).