Evos onstage incubator

A375P cells were seeded in six-well plates (2 × 10⁵ cells/well) and, upon reaching confluence, the monolayer was scratched with a sterile 200 µL pipette tip. A375DR cells were seeded in six-well plates (4 × 10⁵ cells/well) but their monolayer was scratched with a sterile 10 µL pipette tip when they reached confluence. To remove detached cells, wells were washed with complete medium and afterwards refilled with fresh medium containing 10% FBS. 1 µM ONC was added to this culture and samples were compared with the ONC-free ones. A375P cells were incubated for 17 h (1020 min), while A375DR cells for 54 h (3240 min), and the behavior of both cell subpopulations was monitored with EVOS FL Auto Cell Imaging System. Cells were kept at 37 °C in humidified atmosphere with 5% CO₂ in an EVOS Onstage Incubator (ThermoFisher Scientific, Waltham, MA, USA). The A375P cells movement frames were captured every 30 min over the aforementioned 17 h with a 5× magnification, while the A375DR cells frames every 2 h with a 10× magnification over the aforementioned 54 h, to create time-lapse videos. The relative images were quantitatively analyzed with the Image-J computing software and the MRI Wound Healing Tool (NIH, Bethesda, MD, USA). In the graphics, the mean value relative to each point calculated from four independent experiments is reported ± SD.

Cells were plated on 35-mm µ-Dish culture plates (81156, ibidi, USA Inc., Madison, WI, USA) that were coated with laminin 332 matrix [57 (link)], at a density of 1 × 10⁴ cells/cm². Images of cells cultured at 37 °C in a humidified atmosphere containing 5% CO₂ were acquired over a 16-h period, using an EVOS Cell Imaging System equipped with an EVOS onstage incubator (Thermo Fisher Scientific), and capturing a phase-contrast image every 10 min. Image sequences were imported into Fiji software [58 (link)] and the Manual Tracking Plug-in was used to track every cell within each frame. Migration tracks were then analysed using the Chemotaxis and Migration Tool software (ibidi). The migration path of each cell was visually outlined, and the accumulated (total) distance, the euclidean distance (straight linear distance from the initial to the final cell position point) and the average speed of each cell were calculated.

HPSC-cardiomyocytes were seeded as full monolayers (30 K cells per well) onto vitronectin-coated CellCarrier-96 well special optics plates (PerkinElmer) and were treated with 1 μM of the anticancer drugs DOXO, AMR or ACLA 10-12 days after seeding. Images of hPSC-cardiomyocytes were acquired using the high-throughput automated EVOS FL Auto 2 (Thermo Fisher) microscope equipped with a 40x Super-apochromat Olympus objective (NA 0.95) (Thermo Fisher, AMEP4754). Anthracyclines were quantified based on auto-fluorescence using the RFP filter cube (ex531/20nm; em593/40nm, Thermo Fisher, AMEP4652). The cell culture area of the well was scanned by automatically acquiring 55 images per well at indicated timepoints. Cells were maintained during the 24 hours at 37°C and 5% CO₂ on the EVOS FL Auto 2 with the EVOS Onstage incubator (Thermo Fisher). For flow cytometry quantification, cardiomyocytes were treated in suspension with the compounds and autofluorescence was quantified (at ex561nm, em586/15) with a MACSQuant VYB flow cytometer (Miltenyi Biotech) at the indicated time points. Plots were analyzed with FlowLogic software.

Conventional wound healing scratch assays were also used to explore cellular migration rates and were undertaken using an EVOS® FL Auto Imaging System (Life Technologies, CA, USA) equipped with an EVOS® Onstage Incubator (Life Technologies, CA, USA). Cells were prepared in a 24-well plate and grown to form a monolayer before inducing a wound by scratching a 200 µl pipette tip through the monolayer. Following induction of the wound, the monolayer was washed, fresh medium added and cells allowed to recover for 10 minutes before placing the plate in an EVOS® Onstage Incubator pre-set to 37 °C, 5% CO₂ and 80% humidity. Image captures were programmed to three separate areas of each wound and each location was imaged at 1 hour intervals, for up to 20 hours. Wound healing scratch assay experiments involving treatments were undertaken using conventional image capture methodologies using an inverted microscope over 2 hour time points for an 8 hour period. Images of the scratch wound were taken at the same area over the time course and multiple regions of wound closure were quantified within this area. For both methodologies, the distance between the two wound edges was measured using Image J, in pixels, and the distance change at every time interval was calculated through subtraction of the distance between each wound edge at each time point from the wound edge distance at time zero.