Mitotracker orange cm h2tmros

A549 cells were seeded in each compartment of Cellview cell culture dishes (4 compartments, glass bottom, Greiner). The following day, cells were infected with influenza A H1N1 virus at MOI of 1 in a final volume of serum-free DMEM of 250 μl. After 1h incubation at 37°C, the inoculum was removed and 500μl of live microscopy media (DMEM containing no phenol red supplemented with L-glutamine, pyruvate, 10% serum and antibiotics) containing MitoTracker Orange CM-H₂TMRos (ThermoFisher, dilution 1: 10 000) was added. Uninfected control cells were treated in the same way. Image acquisition was performed using a Nikon inverted microscope (EclipseTi2) coupled with a Dragonfly spinning disk unit (Andor, Oxford Instruments Company). An oil immersion objective (100X Plan Apo lambda, numerical aperture (NA) 1.45 0.13mm) was used. Acquisitions were performed every 5 minutes for 15h (3 fields per condition). A z step of 1μm was used for the z-stacks.

OZ2 was amplified from OZ2 cDNA with Phusion polymerase (Thermo Scientific) and the primers OZ2-F1 and OZ2-nostop-R1 using standard protocols. 3′-A overhangs were added with Taq (QIAGEN) by incubating at 37°C for 10 min. After purification, the amplicons were cloned into pCR8/GW/TOPO (Invitrogen) to use in a Gateway cloning reaction with a modified pEXSG vector (17 (link)) containing an EYFP C-terminal tag using LR Clonase II (Invitrogen) to produce pEXSG-OZ2-YFP.
Arabidopsis Col-0 plants and Nicotiana benthamiana were grown on soil in a long day (16 h) conditions for 3–5 weeks for Arabidopsis and 5–6 weeks for N. benthamiana. pEXSG-OZ2-YFP was transfected into Arabidopsis and N. benthamiana protoplasts using the method outlined in (18 (link)), using ∼3.0 × 10⁵ cells per transformation.
Protoplast mitochondria were stained with MitoTracker™ Orange CM-H₂TMRos (500 nM; ThermoScientific), using DMSO as the solvent and W5 buffer. Protoplasts were incubated in the dark for 45 min and then were pelleted (1000 × g, 5 min) and resuspended in W5 buffer (500 μl). Protoplasts were imaged using a Zeiss Axio Observer LSM 710 microscope and C-Apochromat 40×/1.20 W Korr M27 objective.

SkMs were seeded on 24-well plates (2500 cells/well) and treated with free farnesol, farnesol-SUV, farnesol-MLV, control-SUV, or control-MLV, or left untreated. After 24 h, cells were incubated with 1 µM MitoTracker Orange CM-H2TMRos (Thermo Fisher Scientific, Waltham, MA, USA) or 200 nM MitoTracker Red CMXRos (Thermo Fisher Scientific) in DMEM (P04-0359, PAN Biotech, Aidenbach, Germany) for 60 or 30 min, respectively. An automated inverted microscope (DM4000B, Leica Microsystems) with 515–560-nm filter was used to capture the images.

The dissociated cells were loaded with Mitotracker Orange CMH2TM ROS (Thermo Fisher Scientific, Waltham, MA, USA) and incubated on a shaker at 37 °C for 30 min. FACSAria was used (Becton, Dickinson, and Company, Franklin Lakes, NJ, USA) for intracellular ROS measurement.

Mitochondrial membrane potential was measured by FACS analysis of cells stained with MitoTracker® Orange CMH2TMRos (mitochondrial membrane potential evaluation) (Thermo Fisher Scientific, Milan, Italy). WT and GPER KO MDA-MB-231 cells were plated and after seeding they were switched to 1% charcoal-stripped FBS for 36 h to be processed for experiments. Next, the cells were collected and incubated with 10 nM MitoTracker staining solution for 30–60 min at 37 °C. Cells were then harvested, re-suspended in PBS and analyzed by flow cytometry (CytoFLEX Beckman, Beckman-Coulter, Milan, Italy).

Mitophagy signal measurements of CMMR mouse pancreatic sections, ROS measurements using MitoSOX, functional mitochondrial imaging using MitoTracker Orange CM-H₂TMRos (Thermo Fisher Scientific) and immunostaining were performed as described previously^{26 (link)}. To detect apoptotic death of β-cells, pancreatic sections were labeled with a MEBSTAIN Apoptosis TUNEL kit (MBL, Japan) in accordance with the manufacturer’s instructions, followed by staining with anti-insulin antibody (Sigma) and 4′,6-diamidino-2-phenylindole (DAPI). The numbers of TUNEL-labeled nuclei among insulin-positive β-cells were manually counted.