Nc 3000 image cytometer

Image cytometry with an NC-3000 Image Cytometer (ChemoMetec, Allerod, Denmark) was used for cellular and mitochondrial phenotyping. A minimum number of 5000 events were analyzed in each experiment. The following assays were executed according to the manufacturer’s recommendations: cell count (acridine orange and DAPI (ChemoMetec)), mitochondrial membrane potential (JC-1 and DAPI (ChemoMetec)), thiol redox status (VitaBright-48, Propidium iodide, and acridine orange (ChemoMetec)). Mitochondrial superoxide levels (MitoSOX™ (Thermo Scientific) and Hoechst-33342 (ChemoMetec)) were measured as described earlier [43 (link)]. To measure mitochondrial mass, 100 nM fluorescent dye MitoTracker™ Green FM (Invitrogen, Carlsbad, CA, USA) diluted in Hank’s balanced salt solution (HBSS) (Invitrogen) was added to cells for incubation for 30 min at 37 °C. After removing excess dye, the cells were collected and a 1:1000 dilution of RedDot2 (Biotium, Fremont, CA, USA) (concentration not specified) was added to each sample before measurement. The NucleoView NC-3000 software (ChemoMetec) was used for data analysis.

All cytometric fluorescent measurements were performed using the NC-3000 image cytometer (Chemometec) as described in Fernandez-Guerra et al. (2016 (link)). Mitochondrial oxygen consumption rate profiling assays of cultured fibroblasts were performed using a Seahorse XF^e96 extracellular flux analyzer and the Mito Stress test kit (Seahorse Bioscience) as recommended by the supplier with the following modifications: 15,000 cells per well were seeded 24 h before analysis and FCCP was added at 1 μM final concentration. Oxygen consumption rate (OCR) was normalized to total protein amount measured in each well after the analysis using the Bradford Protein assay (Bio-Rad).