Harmony harmony 4.1

To measure cellular proliferation, fibroblasts were plated at 1,000 cells/well in duplicated 96‐well imaging plates 98 h prior to the assay. Twenty‐four hours after plating, one plate was used for imaging of Hoechst staining (1 μg/ml) for cell counting. The second plate was treated with 50 nM (+)‐Epicatechin or DMSO and kept for 72 h until cell counting. Imaging was performed in triplicates with a Perkin Elmer Operetta system (10× objective). Analysis was performed with Perkin Elmer Harmony (Harmony 4.1) software and % change from plate 1 to plate 2 was calculated for the respective cells and treatments.

Mitochondrial ATP content was assessed with the BioTracker™ ATP‐red dye, a live cell red fluorescent imaging probe for ATP. The probe specifically reports ATP content in the mitochondrial matrix of living cells. The probe without ATP forms a closed ring structure that is not fluorescent. In the presence of the negatively charged ATP, the covalent bonds between boron and ribose in the probe are broken and the ring opens, causing the probe to be fluorescent.
Cells were incubated for 1 h with 200 nM MTG and 1 μg/ml Hoechst, washed twice with PBS, and incubated for 15 min with 5 μM BioTracker™ ATP‐red dye (Millipore) in medium at 37°C (5% CO₂).
Before imaging, the cells were washed twice with medium and fresh medium was added. Imaging was performed in triplicates and in two focus planes with a Perkin Elmer Operetta system (20× objective) at 37°C and 5% CO₂. Excitation and emission filters used for the combination of dyes: Hoechst (ex. 360–400, em. 410–480), MTG (ex. 460–490, em. 500–550) and mtATP Red (ex. 560–580, em. 590–640). Analysis was performed with Perkin Elmer Harmony (Harmony 4.1) software by measuring the average ATP red fluorescence intensity inside a region of interest (Filimonov et al, 2014 ) generated by the MTG area.

Cells were incubated for 1 h with 200 nM MTG, 200 nM TMRE (Biotium) and 1 μg/ml Hoeschst, washed twice with PBS, and incubated with 200 nM in medium at 37°C (5% CO₂). Imaging was performed in triplicates and in two focus planes with a PerkinElmer Operetta CLS high‐content system (20× objective) at 37°C and 5% CO₂. Analysis was performed with Perkin Elmer Harmony (Harmony 4.1) software by measuring mean TMRE fluorescence intensity inside an ROI generated by MTG area.