Leica af 6000 epifluorescence microscope

For mitochondrial potential measurements, hPSC-CMs plated in individual petridishes with glass bottoms (35 mm) were used 1 day after the medium change. Tetramethylrhodamine (TMRE; 1200 nM; T669, ThermoFisher (Waltham, MA, USA)) and Mitotracker Green (MTG; 100 nM; M7514; ThermoFisher (Waltham, MA, USA)) were added to the culture media and incubated for 30 min at 37 °C, 5% CO₂. Hereafter, the hPSC-CMs were washed 3 times with CDM3. A time-lapse (200 pictures in 12 min) was made using with a Leica AF-6000 epifluorescence microscope (Leica (Wetzlar, Germany)). After 1 min, Rotenone (4 µM) and Antomycin-A (2 µM) were added to the CDM3 medium. One min before the end of the time-lapse, (20 µM) were added. Imaging analyses were performed using ImageJ.
For Mitochondrial calcium release, hPSC-CMs plated in individual Petri dishes with glass bottoms (35 mm) were used 1 day after the medium change. Fluo4-AM (5 µM; F14201; ThermoFisher (Waltham, MA, USA)) and Thapsigargine (10 µM; BML-PE180-001; Enzo life sciences (Bruxelles, Belgium) were added to the culture media and incubated for 230 min at 37 °C, 5% CO₂. Hereafter, the hPSC-CMs were washed 3 times with Ca²⁺-free Krebs buffer. A time-lapse (200 pictures in 30 min) was made using with a Leica AF-6000 microscope. After 2 min, FCCP (20 µM) suspended in Krebs buffer was added. Imaging analyses were performed using ImageJ.

The JC‐1 assay kit (Beyotime) was employed to evaluate the alteration in the mitochondrial membrane potential (MMP, △Ψm) by measuring the change in the JC‐1 levels. JC‐1 selectively enters mitochondria, exhibiting different fluorescence properties in accordance with changes in MMP. With elevated MMP, JC‐1 polymerizes and exhibits a crimson luminescence. While with lower MMP, it exists as a monomer and fluoresces green. The cells were subjected to QDN concentrations for a period of 48 hours. Cells were then examined and visualized with an inverted Leica AF6000 epifluorescence microscope (Leica).