Mitotracker red

Cells were pre-treated with or without 10 μM spermidine, 5 mM NAC, or 5 μM BAPTA-AM for 1 h before incubation for 24 h in the presence or absence of 300 μM H₂O₂. To assess the function of mitochondria and the endoplasmic reticulum, we stained cells with 100 nM MitoTracker Red and 1 μM ER-Tracker Red probe, respectively, before observation using a fluorescence microscope (Carl Zeiss) per the manufacturer’s instructions.

To evaluate mitochondrial morphology, living RPTECs were incubated with 250 nM fluorescent probe MitoTracker Red (#M7512; Thermo Fisher, Invitrogen) for 30 min, at 37 °C, 5% CO₂. Nuclei were counterstained with NucBlue Live ReadyProbes (Hoechst; #R37605; Thermo Fisher, Invitrogen) according to the manufacturer’s protocol. At the end of probes incubations, living cells were examined by confocal inverted laser microscopy (LSM 510 Meta, Zeiss) and mitochondrial fragmentation was expressed as the % of Hoechst positive cells with fragmented mitochondria compared to elongate one identified by MitoTracker Red staining in 10 random fields per sample.
Mitochondria membrane potential was evaluated by exposing RPTECs to 5 μM JC-1 (#T3168; Thermo Fisher, Invitrogen) for 30 min, at 37 °C, 5% CO₂. JC-1 exhibits potential-dependent accumulation in mitochondria indicated by a fluorescence shift from green (cytoplasm) to red (mitochondria). At the end of probe incubations, living cells were examined by confocal inverted laser microscopy (LSM 510 Meta, Zeiss) and the quantification of JC-1 red and green areas (Pixel^{2 (link)}; Image J 1.40g software) was performed in 10 random fields per sample and mitochondria polarization was expressed as the ratio between red and green fluorescent area.

The animals were incubated for 4 h at 20 °C with 10 μM MitoTracker Red (Invitrogen, Carlsbad, CA, USA), which is a fluorescent probe that accumulates in active mitochondria. After MitoTracker staining, the animals were immobilized in 10 µL of M9 buffer containing 100 µg/mL tetramisole on a poly L-lysine-coated slide, and the animals were dissected for oocyte isolation. Live images of stained animals were observed under a fluorescence microscope (Zeiss Axioscope, Oberkochen, Germany), and the average pixel intensity of MitoTracker Red fluorescence was measured using ImageJ software. To measure MMP, tetramethylrhodamine methyl ester (TMRM; Thermo Fisher Scientific, Waltham, MA, USA) staining was performed, as previously described [30 (link)]. Briefly, TMRM was added to NGM agar at a final concentration of 30 μM, and the plates were dried overnight and seeded with E. coli OP50 for 24 h in the dark. Animals from each group (young, aged, and NAM+aged) were transferred to TMRM plates, incubated at 20 °C for 15 h, and dissected for oocyte isolation. For whole-body animal staining, each group of mothers was incubated in TMRM plates for 15 h. Images were obtained under the same exposure, and the fluorescence intensity was measured using ImageJ software by selecting oocytes and whole animals.