Mitotracker red fm

Real-time changes in cellular (cytoplasmic) NADH content were estimated by measuring endogenous NADH fluorescence with 360 nm excitation and > 410 nm emission^{59 (link)}. Fluorescence images were acquired at 1 min intervals after 5 min of baseline recording. Regions of interest were selected from the nucleus to eliminate signal from mitochondria, and cytochalasin D (1 µm) was included to prevent cell movement. Images acquired from each cell were used to calculate change in fluorescence intensity / average of pre-treatment fluorescence intensity (ΔF/F_o). In a subset of experiments cells were pre-incubated with 200 nm Mitotracker Red-FM (Molecular Probes) to verify that the NADH fluorescence measurements were not contaminated by mitochondrial NADH fluorescence. Mitochondrial and NADH signals were imaged by interleaving NADH fluorescence excitation and Mitotraker (excitation 550 nm; emission > 610 nm).

Baby green monkey kidney (BGMK) was obtained from the American Type Culture Collection (ATCC; Manassas, VA). BGMK cells were cultured in Dulbecco-modified Eagle’s medium (DMEM; HyClone, Ottawa, ON) with 7 % fetal bovine serum (FBS; HyClone), 2-mM L-glutamine, 100-U/mL penicillin, and 100-μg/mL streptomycin, at 37 °C with 5 % CO2. The following antibodies/probes were used during immunofluorescence: Lysotracker®DND-99 from Molecular Probes (Burlington, ON), the 9E10 mouse and mouse myc monoclonal antibody from Roche (dilution 1/100; Indianapolis, IN), rabbit anti-FLAG (dilution 1/100; Burlington, ON), and FITC/Cy3-conjugated goat anti-mouse or anti-rabbit immunoglobulin G (IgG) from Jackson ImmunoResearch Inc. (dilutions 1/100, 1/200 respectively; West Grove, PA, anti-CD63 antibody from Invitrogen (dilution 1/100; Burlington, ON); and MitoTRACKER Red FM from Molecular Probes (Burlington, ON). LITAF WT was synthesized by GenScript (Piscataway, NJ). Myc-tag was added to the N-terminus of the protein to facilitate imaging. PMP22 was synthesized by Sino Biological Inc. (BC019040, Beijing). The gene was pCMV/hygro and contained a flag-tag.

HepG2 and Huh7 cells were plated on 18-mm cover glasses for 24 h and then treated with LAC117 (100 μg/mL). A mitochondrion-specific dye (MitoTracker Red FM: Molecular Probes Inc., Eugene, OR, cat.n.M22426) was added and incubated for another 30 min. The media were removed, and the cells were washed with PBS and fixed with an acetone: methanol solution for 5 min at − 20 °C. The fixed cells were washed with PBS for several times and incubated with cytochrome c antibody (Santa Cruz Biotechnologies, cat.n.13156) overnight at 4 °C. Subsequently, after washing with PBS several times, the cells were incubated with a mouse fluorescenct-labeled secondary antibody (1:100, Vector Laboratories, Burlingame, CA, cat.n.TI-2000) for 1 h at room temperature. The cells were stained with DAPI to visualize the nuclei. Finally, the cells were covered with a fluorescent mounting solution (Dako, Carpinteria, CA, cat.n.REF3023) before viewing with a confocal laser scanning microscope (Olympus, Tokyo, Japan).

KPL-4 cells were grown on LabTek-II eight-well slides to approximately 70% confluency and incubated with 2 µM MC1_Cy5.5 and the micropinocytic probe Oregon Green Dextran (Molecular Probes, D7171; 200 µg ml⁻¹) and/or the clathrin-dependent recycling marker Alexa 555–transferrin (12.5 µg ml^–1; Molecular Probes, T35352) for different lengths of time. Cells were then washed four times in cold medium, fixed for 20 min in 3% paraformaldehyde, washed twice for 5 min each in PBS and mounted in Prolong Gold plus DAPI (Invitrogen, P36931). Mitochondria were stained with 200 nM Mitotracker Red FM (Molecular Probes, M22425) for at least 20 min before live imaging.
Spinning disk confocal microscopy was performed using a CSU-W1 (Yokogawa) spinning disk on a Zeiss AxioObserver M1 microscope with a ×63/1.4-NA objective equipped with 405-, 488-, 561- and 640-nm lasers. Images were acquired using SlideBook v6 (Intelligent Imaging Innovations) and a Photometrics Prime BSI (Teledyne Photometrics). Figures were assembled in Adobe Photoshop 2021, and any gamma adjustments to the contrast were applied across the whole image.

Granulosa cells were revealed by positive staining of the follicle-stimulating hormone receptor (FSHR). Granulosa cells were treated with a rabbit anti-FSHR primary antibody (cat# 3929, Sigma-Aldrich, Saint Louis, MO, USA) and then a goat anti-rabbit immunoglobulin G (IgG) secondary antibody (cat# ab6717, Abcam, Cambridge, UK).
For the analysis of mitochondrial membrane potential, granulosa cells were concurrently treated with N, N, N’, N’-tetra-methylethylenediamine (TMRE) (cat# T669, Molecular probes, Life Technologies, Auckland, NZ) or MitoTracker Red FM (cat# M22425, Molecular probes) at 37 °C for 60 min, washed with PBS and centrifuged at 250 g for 15 min at room temperature to remove excess dye. For the analysis of mitochondrial mass, granulosa cells were stained with nonyl acridine orange (NAO) (cat# A1372 Molecular probes) at 37 °C for 30 min. Following staining and washing with PBS, the samples were centrifuged at 250 g for 15 min at room temperature to remove excess dye.
Flow cytometry analysis was performed using a FACSCalibur system (Becton, Dickinson and Company, BD Biosciences; USA) with the side-scattered light (SSC) detector voltage set at 415, the FL1 detector voltage set at 635, the FL2 detector voltage set at 614, and the FL3 detector voltage set at 150 voltage.