4.9. Assessment of Mitochondrial Activity

The mitochondrial activity in bovine granulosa cells transfected with miRNA, siRNA and Que was assessed using MitoTracker^® Red CMXRos (M7512; Invitrogen) according to manufacturer′s instructions. Breifly, lyophilized MitoTracker^® product was dissolved in a high-quality, anhydrous dimethylsulfoxide (DMSO) to a final concentration of 1 mM. The final working concentration (200 nM) was prepared by diluting stock solution in DMEM/F12-HAM medium. MitoTracker^® Red CMXRos is a red-fluorescent dye that stains mitochondria in live cells and its accumulation is dependent upon membrane potential (MMP), which is a marker for mitochondrial functionality [114,115]. For that, granulosa cells were cultured in eight-well slide and cells were incubated with 200 nM MitoTracker^® red dye at 37 °C for 30 min, followed by two washing with PBS-CMF and then fixed overnight at 4 °C with 4% paraformaldehyde. Fixed cells were mounted with Vectashield (H-1200) containing DAPI. Images were acquired at 40x magnification under a CLSM LSM-780 confocal laser-scanning microscope (Carl Zeiss GmbH; Germany) and analyzed using ImageJ 1.48v (National institutes of Health, Maryland, USA, https://imagej.nih.gov/).