Mitotracker green fm

MSCs were stained with MitoTracker Green FM (Invitrogen, Carlsbad CA, USA) to quantify functional mitochondrial mass. Briefly, 48 h prior to staining 1 × 10⁴ cells were seeded using 35 mm glass bottom culture dishes, containing 14 mm microwell (MatTek, MA, USA). Cells were incubated for 30 min with DMEM supplemented with 80 nM MitoTracker Green FM, followed by 5 μM CellTracker Red CMTPX solutions to stain the cytoplasm (Invitrogen), and Hoechst (Invitrogen) to define cell nucleus. Cells were observed in vivo, and 2D and 3D images were collected by confocal microscopy using an Olympus FV1000 with an excitation/emission range of 400/545 for MitoTracker Green FM and 577/602 nm for CellTracker Red CMTPX. Z-stack parameters were as follows: ~15 z-axis slices at ~0.50 μm intervals with a final Z-stack thickness of ~7.5 μm. Mitochondrial mass was determined by data obtained from confocal microscopy in voxel units.

Spermatozoa collected from the cauda epididymis (from mice at age of 8–16 weeks) were cultured for 2.5 h in mHTF medium, with or without 10 µM CGP37157 (Cayman) for the last 60 min and with 0.1 μM Rhod2-AM (Abcam) and 0.1 μM MitoTracker Green FM (Thermo Fisher Scientific) for the last 30 min on a glass-bottom dish coated with laminin. The mHTF containing Rhod2-AM and MitoTracker Green FM was replaced with mHTF containing DAPI (6 μg/ml), and the cells were observed with an Olympus IX70 microscope. The quantification of fluorescence values was performed with the use of ImageJ.