Mitotracker cm h2xros

Mitochondrial ROS levels were determined using MitoTracker CM-H2XRos (Invitrogen Life Technologies, Carlsbad, CA, USA), as previously described [36 (link)]. RA-differentiated SH-SY5Y cells were seeded onto coverslips and subjected to the experimental protocol. Subsequently, the cells were loaded with a dye concentration of 300 nM for a duration of 30 min at 37 °C. Confocal images were obtained using a 510 LSM microscope (Carl Zeiss, Milan, Italy) equipped with a META detection system. For visualization of MitoTracker CM-H2XRos, the dye was excited at 560 ± 10 nm, and its emission was measured at 620 ± 20 nm. Images were captured at 5 s intervals, allowing for the monitoring of basal ROS levels over approximately 200 s. Following image acquisition, the fluorescence intensity was analyzed offline. The fluorescence values were expressed as a percentage relative to the control value.

ROS production from mitochondria was visualized using the mitochondrial-targeted dye MitoTracker CM-H2XRos [74 (link)] (Invitrogen Life Technologies, Carlsbad, CA, USA). Cells grown on glass coverslips (coated with poly-D-lysine for neuronal cultures) were treated according to the protocol routines and then loaded with 300 nM of the dye for 30 min, at 37 °C, in the dark. CM-H2XRos was excited at 561 nm, and fluorescence emission was collected above 580 nm and recorded for ~60 s using a confocal microscope Zeiss 510 LSM (Carl Zeiss, Milan, Italy). Changes in the red fluorescence were analyzed offline after image acquisition. Fluorescence values were reported as percentages of the control value.

Mitochondrial ROS production was evaluated using the mitochondrial-targeted dye Mitotracker CM-H2XRos [55 (link)] (Invitrogen Life Technologies, Carlsbad, CA, USA). RA-differentiated cells were cultured on glass coverslip on 6-well plates (1 × 10⁵ cells/well) and, after being treated, cells were loaded with 300 nM of the dye for 30 min in the dark at 37 °C. For live cell imaging of mitochondrial ROS, measurements were conducted on the confocal microscope Zeiss 510 LSM (Carl Zeiss, Milan, Italy). CM-H2XRos was excited with 561 nm laser, and emission was detected above 580 nm and recorded for 120 s. The analysis of red fluorescence increase was performed off-line after images acquisition. Fluorescence values were reported as percentage of the control.

To analyze the filamentation capability of U. maydis in PD-charcoal plates, single colonies where visualized using the Leica M205 Stereoscope equipped with an ORCA-Flash4.0 LT Hamamatsu digital camera. The area of the colonies was measured by selecting the perimeter of each colony using the plugging convex hull of ImageJ software. To determine sirtuins’ localization, sir2:eGFP, hst2:eGFP, hst4:eGFP, hst5:eGFP and hst6:eGFP cells were visualized using a DeltaVision microscopy system comprising an Olympus IX71 microscope and CoolSnap HQ camera (Applied Precision, Issaquah WA, United States). To visualize mitochondria, 0.5 mM Mito-Tracker CM-H2Xros (Molecular Probes, Eugene, OR) was added to the U. maydis YEPSL cell culture and cells were incubated for 15 min at 25°C (Bortfeld et al., 2004 (link)). To analyze the U. maydis progression inside the maize plant, leaves samples from 3, 4 and 6 dpi infected plants were distained with ethanol, treated for 4 h at 60°C with 10% KOH, washed in phosphate buffer and then stained with propidium iodide (PI) to visualize plant tissues in red and wheat germ agglutinin (WGA)/AF488 to visualize the fungus in green. At least four leaves from two independent experiments were stained and visualized by fluorescence microscopy (Leica SPE DM2500, Leica, WZ, Germany). Image processing was carried out using the ImageJ software.

Pro- and antioxidant activities were evaluated using 2′,7′-diidrodiclorofluoresceina diacetate H2-DCFH-DA probes. Hepatocytes cells were plated in a multi-well and incubated for 24 h at 37 °C and 5% CO₂. After that, cells were washed with 7.4 phosphate buffer saline (PBS) and treated with H2-DCFH-DA (100 µM as a final concentration) in RPMI for 30 min, at 37 °C and 5% CO_2.The cells were then washed again with PBS and treated for 6 h with the different compounds in the exam. Fluorescence was measured on the automated plate reader NOVOstar (BMG Labtech, Ortenberg, Germania), using an excitation wavelength of 485 nm and an emission wavelength of 530 nm. In the same way, the cells were treated to determine the ROS mitochondrial. In this case, MitoTracker CM-H2XROS (Molecular Probes/Invitrogen, Waltham, MA, US) was used as a probe, using an excitation wavelength of 543 nm and an emission wavelength of 590 nm.