Mitotracker green fm dye

Mitophagy was studied by measuring the colocalization of mitochondria and lysosomes (autophagolysosome formation). Mitochondria were stained with MitoTracker™ Green FM dye (m7514, Thermo Fisher, Villebon-sur-Yvette, France) at a concentration of 100 nM. The staining of lysosomes was performed by using the 60 nM of LysoTracker™ Red DND99 (Invitrogen, Illkirch, France). Images were observed using fluorescence microscopy (Nikon eclipse Ti, Okolab, Rome, Italy) and Nikon’s NIS-Elements microscope imaging software. Pearson’s correlation coefficient (r) has been used for quantifying colocalization using the JACOP plugin for ImageJ. Autophagolysosome formation was studied following treatments with 30 µM DOX (Sigma, Quentin Fallavier, France) for 72 h or the combination of DOX and 1 µM mitoTEMPO (Sigma, Quentin Fallavier, France) for 24 h.

ROS was measured using CellROX Deep Red Reagent (Invitrogen) and MitoTracker Green FM Dye (Invitrogen) (Lagadinou et al., 2013 (link); Minai et al., 2013 (link)). Briefly, cells were co-cultured for 24 hr followed by loading with CellROX dye (5 mM) and MitoTracker Green dye (100 nM) at 37°C for 30 min, then analyzed by flow cytometry. The data were analyzed using Flowjo software (Tree Star Inc, Ashland, OR).

Mitochondrial mass of cultured PO-MSCs was quantified by flow cytometry. Briefly, 1 × 10⁵ cells were stained in PBS with MitoTracker Green FM dye (Invitrogen, Waltham, MA, USA) for 30 min, washed, and resuspended in 200 μL PBS with 1% fetal bovine serum. Cellular fluorescence signals were then analyzed using a FACSCalibur (BD Biosciences, San Jose, CA, USA). Resulting flow cytometry data were analyzed using the FlowJo software version 8.7.3 (Ashland, OR, USA). For fluorescent microscopy, PO-MSCs cultured on chamber slides were stained with MitoTracker Green FM dye. Cellular mitochondria were visualized under a fluorescent microscope (Zeiss Axio Observer Z1, Carl Zeiss, Oberkochen, Germany), and images were analyzed using the ImageJ software (NIH, Bethesda, CA, USA).

Mitochondrial activity was measured by Mitotracker Green FM. In short, BT549 and Hs578T parent cells, and the BT549 and Hs578T with or without ATM were seeded at 1.5 × 10⁵ cells/well. After culture under normoxia for 24 h, parent cells were treated with 10 μM of KU60019 and incubated in 1% O₂ for around 12 h, and then 10 nM of MitoTracker Green FM dye (Invitrogen) were added to all cells and continuously incubated for 1 h at 37 °C before dying. Cells were collected by trypsinization, washed, and re-suspended in phosphate-buffered saline, followed by flow cytometry. The data were analyzed using FlowJo software. All experiments were performed at least three times.

The hCECs (1 × 10⁵ cell per well) were seeded and cultured in SHEM in a 6-well plate for 24 h. The cells were then treated with prednisolone or solvent and further cultured for 3 days. For apoptosis assay, the cells were stained with a combination of PI and Annexin V (Cat no. 556547, FITC Annexin V Apoptosis Detection Kit I, BD Pharmingen™, San Diego, CA) at room temperature for 5 min and analyzed by flow cytometry (S1000EXi Flow Cytometer, Stratedigm, Inc., San Jose, CA). For mitochondrial ROS measurements, the cells were stained with both CellROX dye (5 μM, CellROX^TM Deep Red Reagent, Invitrogen) and MitoTracker Green dye (100 nM, MitoTracker Green FM Dye, Invitrogen) at 37 °C for 30 min, and analyzed for fluorescence using flow cytometry. The data obtained from flow cytometer were analyzed using Flowjo software (Tree Star, Ashland, OR).

The Mitochondrial mass was measured by a MitoTracker Green FM dye (Invitrogen), a dye that stains mitonchondria independent of its membrane potential. Cells were stained with 100 nM MitoTracker probes at 37 °C for 30 min and washed two times with PBS. The fluorescence of MitoTracker (excitation 495 nm, emission 520 nm) was measured at 25 °C using a BioTek Synergy microplate reader (BioTek Instruments, Winooski, VT).