C3100mp

Mouse embryonic fibroblast NIH/3T3 cells (ATCC, CRL-1658) were seeded on 48-well plates, with a density of 20,000 cells per well, in Dulbecco’s Modified Eagle Medium (DMEM, Gibco 12,800–017), supplemented with 10% fetal bovine serum (SFB; Capricorn Scientific FBS-HI-11A) and 1% penicillin G-streptomycin (Gibco 15,140–122) at 37 °C under a humid atmosphere at 5% CO₂. Once 80% confluence was reached, cells were treated with 10, 100, and 500 μg/mL of each CD’s formulation for 24 h. After treatment, cells were incubated with 2 µM Calcein-AM (Invitrogen C3100MP) and 1.5 µM propidium iodide (PI, Sigma P4170) in DMEM without phenol red for 30 min at 37 °C. Calcein-positive cells, stained in green, correspond to live cells, while red nuclei are dead cells. Cells were immediately imaged using a 20 × objective with a Dmi8 Leica fluorescence microscope and analyzed by automatic counting with the Analyze Particles tool of the ImageJ software (NIH, USA). Viability was calculated as the percentage of live cells normalized against the total cell number (calcein + PI cells). Plots and statistical analysis were performed with the GraphPad Prism software v8.0, with results obtained from n = 4 independent experiments.

HUVECs (1.6 × 10⁴ cells/cm²) (Sigma, 200P-05N) and INS-1 832/13 cells (2.6 × 10⁴ cells/cm²)^{36 (link)} were plated on 24-well plates with either no coating, collagen I coating (Corning, 354249) or hP-HG coating, and cultured for 1–3 days. Cells were stained with Calcein-AM (ThermoFisher, C3100MP) and ethidium homodimer (Sigma, 46043-1MG-F) in a live/dead assay: Samples were imaged with a Zeiss Axiovert microscope; 10X images were used to count live cells (green) and dead cells (red) using Image J software. Each cell type was tested with 2 biological replicates of 4 technical replicates each.

mPTP opening was assayed by measuring calcein (Molecular Probes, #C3100MP) fluorescence quenched by cobalt chloride (Sigma, #60818), as previously reported. In brief, H9C2 cells were loaded with 2 μM calcein/AM and 2 mM CoCl₂ at 37 °C for 30 min. After washing, cells were illuminated at 488 nm and an emission wavelength of 525 nm was captured with the Enspire Multimode Plate Reader every 30 s. Results were expressed as a percentage of normoxic group.