Zoom software

HeyA8-MDR/HeyA8, SKOV3-TR/SKOV3-TR (3 × 10³) cells were seeded in a 96-well plate. Scratches were generated using a 96-pin wound maker (Essen BioScience, Ann Arbor, MI) and cells were treated with free tariquidar (XR), paclitaxel (PCT), LP(XR), LP(PCT), or LP(XR,PCT) (50 nM, PCT; 40 nM, XR) or not treated. The cells were imaged in real-time every 2 hrs using an IncuCyte^® system (Essen BioScience) with phase-contrast microscopy at a 10X magnification (42 (link), 43 (link)). Changes in confluence of the wound/scratch were quantified by Zoom software (Essen BioScience).

5×10⁴ GFP or RFP-labeled tumor cells were cocultured with 5×10⁴ GFP CAR-T cells in 200μL RPMI supplemented with 10% FBS, 10mM HEPES, 2mM L-glutamine, 100 U/mL penicillin, and 100μg/mL streptomycin. Triplicate wells were plated in 96-well flat-bottom plates for each condition. Tumor fluorescence was monitored every 2–3 hours with a 10x objective using the Incucyte Zoom system (Essen Bioscience), housed in a cell culture incubator at 37°C and 5% CO₂, set to take 4 images per well at each time point. Total integrated GFP or RFP intensity was quantified using the Zoom software (Essen Bioscience). Data were normalized to the first timepoint and plotted as fold change in tumor fluorescence over time. For cytokine secretion analysis, cocultures were setup as above except in 96-well round bottom plates. After approximately 24 hours, plates were spun down to pellet cells and 100uL of supernatant was harvested and stored at −80°C until analysis. IFNγ and IL-2 levels in coculture supernatants were quantified by ELISA (Human ELISA MAX Deluxe, Biolegend) according to the manufacturer’s instructions. Negative cytokine values were set to 0. Coculture experiments were setup using day 10 T cells, with the exception of On/Off kinetics experiments in Fig. 1G and S1F, which were setup on Day 14.

HUVECs were seeded at a density of 50,000 cells/well into a 96-well ImageLockTM tissue culture plate (Essen BioScience) and incubated in MV2 medium overnight followed by standardized scratching using WoundMakerTM (Essen BioScience). Cells were washed with PBS, and the plate was placed into an IncuCyte ZOOM (Essen BioScience) and scanned every 15 min for 12 h using a 10× objective, during which data were collected using ZOOM software (Essen BioScience). Cellular migration was analyzed using MTrackJ in ImageJ by manually marking the cells at each time frame. The migration speed of one cell was obtained by dividing the migration distance by time.