Sd osr

Fibroblasts cultured on 35 mm glass plates (World Precision Instruments) to 50% confluence were co-stained with MitoSOX Red (ThermoFisher Scientific, cat. M36008) and MitoTracker Green (ThermoFisher Scientific, cat. M7514). Fresh fibroblast medium (2 mL) containing 5 μM MitoSOX Red and 70 nM MitoTracker Green was added to the cells and incubated for 20 min at 37°C. Cells were then washed with DPBS and new medium was added. Cells were incubated at 37°C for 20 min for de-staining and then imaged using an Olympus spinning disc confocal system (Olympus SD OSR) operated using Metamorph software. Cells were then analyzed using ImageJ. Briefly, each cell was isolated through a selection tool on both treatment images. Once identified, remaining fluorescence in the image was cleared. The cells were then subjected to a defined threshold to keep brightness consistent. Both channels were then combined using the image calculator resulting in a cell expressing co-localized fluorescence. Mean gray intensity of the cells was then calculated and plotted using GraphPad Prism 7. Seventy individual cells were quantified per cell line and treatment. Significance was determined through a one-way ANOVA with a Holm-Sidak correction for multiple comparisons.