Fluo4 am calcium dye

In order to assess the intracellular calcium increase, cells were treated for 24 h with STC, TCZ, CsH, or rIL-6. The next day, cells were treated for an additional hour, washed with Hanks’ Balanced Salt Solution (HBSS), and incubated with a mixture of the Fluo4 AM calcium dye and Pluronic acid (Thermo Fisher Scientific) for 30 min at room temperature (RT) in the dark. Cells were washed twice with HBSS to remove the unloaded dye. A standard curve of calcium was prepared in HBSS buffer without calcium. The cytosolic calcium in samples was measured by using a fluorescence microplate reader (Molecular Devices, Menlo Park, CA, USA) using excitation.

HiPSC-cardiomyocytes were seeded as monolayers onto vitronectin-coated CellCarrier-96 well special optics plates (PerkinElmer) in TDI medium and treated with the anthracyclines 10-12 days after seeding at the indicated concentrations. To quantify calcium kinetics and accumulation, cardiomyocytes were stained with the Fluo4-AM calcium dye (Thermo Fisher Scientific) at a final concentration of 1 μM with Pluronic (0.2 mg/ml) for 30 min at 37°C. Imaging was performed 24 hours or 2 weeks after treatment at 37°C and 5% CO₂ on a Nikon Eclipse TE2000-U Microscope with a 20x objective. Videos were taken at 70 fps, which were used to quantify Ca²⁺ fluxes into and out of the cell over time using a custom software script. The individual frame at the lowest fluorescent intensity (= complete relaxation of the cells) was used to determine the baseline Ca²⁺ load of the cardiomyocytes after drug treatment by measuring mean fluorescent intensity of the entire frame using ImageJ software.

The CM-H2DCFDA dye, Fluo-4 AM calcium dye (Molecular Probes, United States), and JC-1 dye (Molecular Probes, United States) were employed to assess ROS, calcium, and MMP, respectively. The fluorescent images of calcium and JC-1 were captured by a confocal microscope. H9c2 cells (1 × 10⁴/well) were seeded in μ-Slide 8-well glass bottom plates. Cells were labeled by Fluo-4 AM or JC-1 probe for 30 min after LS102 treatment according to the manufacturer’s protocols. The fluorescent intensity of images was analyzed by using the Image J software and a randomly captured method was used to make sure there were three fields per group. The fluorescence intensity of calcium, ROS were detected by the flow cytometer. All cells in the 6-well plates were collected and then suspended in a pre-warmed DMEM buffer containing CM-H2DCFDA or Fluo-4 AM calcium dye. Each group (1 × 10⁴ cells) intensity was calculated as representation.