Mitospy orange cmtmros

To mark the endoplasmic reticulum (ER), βTC3 and INS1E cells were transfected with an ER-GFP construct (GFP fused to the ER retention signal of calreticulin, a kind gift of Dr. Piccoli Giovanni, University of Trento, Italy) using Lipofectamine 3000 (L3000015, Invitrogen). Mitochondria were labeled with 100 nM MitoSpy^TM Orange CMTMRos (424803, Biolegend) by following the procedure described above. At 48 h after transfection cells were fixed using 4% PFA (paraformaldehyde) and random fields were captured using the 488 nm and the rhodamin filters of the Axio Observer Z1 microscope (Zeiss). To enhance the image quality prior to the colocalization analysis, the background was substracted and images were pre-processed using the “unsharp mask” filter. Images colocalization was evaluated by means of the ImageProPlus software.

βtc3 cells were pre-loaded with 100 nM MitoSpy™ Orange CMTMRos (BioLegend, 424803, Campoverde Srl, Milan, Italy) in 11 mM glucose Krebs–Ringer buffer at 37 °C for 30 min. Samples were positioned in an imaging chamber and random fields were imaged by using the rhodan filter of the Axio Observer Z1 microscope (Zeiss, Oberkochen Germany). To evaluate mitochondrial morphology, the following parameters were analyzed by using the ImageJ particle analyzer software: area (μm²), circularity (4πArea²/Perimeter²) and Feret’s maximum diameter (μm) [37 (link)].
For time-lapse experiments, single-cell imaging was carried out at 1 frame per second for 30 s under control or oxidative stress conditions. To measure the mitochondrial cumulative distance (µm²), images were first corrected for photo bleaching, then videos were analyzed by using an existing Image-Pro Plus Plug-in (object tracking) (Media Cybernetics, Rockville, MD, USA). Up to twelve cells were imaged in three independent experiments and data were presented as mean values and standard deviations.

The mitochondrial membrane potential and mitochondrial mass of individual cells were determined using MitoSpy Orange CMTMRos (424803; BioLegend, San Diego, CA, USA), which is useful to indicate cell health and mitochondrial localization, and MitoSpy Green FM (424805, BioLegend), respectively. HEK293 cells (2 × 10⁴/mL) were passaged on glass bottom confocal dishes (30012, SPL), treated with microtubule stabilizers (1 nM Taxol and 2 pM EpD) or a microtubule disturber (10 nM VNB) for 16 h; incubated in DMEM containing 250 nM MitoSpy Green FM and 250 nM MitoSpy Orange CMTMRos for 5 min; and washed with culture medium. Then, nuclei were stained with 1 µg/mL Hoechst 33342^® (H1399, Thermo Fisher Life Technologies) for 5 min at room temperature. Finally, samples were imaged using a confocal microscope with a live-cell chamber system (LSM880, Carl Zeiss AG). For time-lapse live-cell imaging, 120 images were captured at a time interval of 0.5 s. Each image was analyzed and exported as a moving file (25 frames/s) and a single picture (TIFF format) using ZEN2012 software (Carl Zeiss AG). Images were evaluated using ImageJ (version 1.08) with the Difference Tracker plug-in. The movies were used to calculate the average percentage of mitochondrial intensity moving and maximum speed of mitochondria in the cytoplasm.

βtc3 cells and human islets of Langerhans were incubated with 100 nM MitoSpy™ Orange CMTMRos (BioLegend, 424803, Campoverde Srl, Milan, Italy) or 100 μM MitoSpy™ Green FM (BioLegend, 424805, Campoverde Srl, Milan, Italy) for 30 min at 37 °C; fluorescence intensities were detected with the microplate reader TECAN Infinite^® F500 (551/576 nm Ex/Em for MitoSpy™ Orange CMTMRos; 490/516 nm Ex/Em for MitoSpy™ Green) (TECAN Infinite^® F500, Tecan Group Ltd. Männedorf, Switzerland). Cells were incubated with 500 μM H₂O₂ for 20 min and fluorescence intensity was detected as previously described. Mean values and standard deviations were based on three different experiments.

MitoTracker-Green (MTG, C1048; Beyotime Biotechnology) is an MMP-independent dye that can monitor the mitochondria. After treatment, the cells were stained with MTG (100 nM) and analyzed using flow cytometry. Mean fluorescence intensity (MFI) was quantified using the FlowJo software (Tree Star, San Jose, CA, USA).
MitoSpy Orange CMTMRos (MitoSpy, 424803; BioLegend) is a cell-permeant, fluorogenic chemical reagent that is used to label the mitochondria in living cells. After the cells were stained with MitoSpy (50 nM), laser scanning confocal microscopy (LSCM; Olympus, Tokyo, Japan) was used to observe the mitochondrial morphology.