Nis element analysis software

After initially seeding, both cell lineages were allowed to grow and attach the wall for 24 hours. After 24 hours, we starting to treat cells with cocktailed stimulators for 10 days, and we counted this initial treating day as day 0. Subsequently, cellular morphological alterations were recorded and evaluated at days 1, 2, 3, 4, 5, 6 and 7 by an inverted microscope Nikon (Nikon) and NIS‐Element analysis software (Nikon) for image analysis. TRAP staining kits were purchased from Sigma‐Aldrich Co. According to instructions, after PBS washing two cell lineages were fixed by paraformaldehyde (4%) lasting for 30 minutes. Subsequently, both cell lineages were washed by PBS and then stained by TRAP solution at 37°C in 1 hour, the staining results were evaluated by Nikon (Nikon) and NIS‐Element analysis software (Nikon) for image analysis.

Time-lapse experiments were performed utilizing a Yokogawa spinning Disk (ECLIPSE Ti2-E) confocal microscope with W1-SoRa module (Nikon). 30 hpf embryos were anesthetized in 0.016% tricaine solution and included laterally in 1% low-melting point agarose in 35 mm confocal Petri dishes to provide stability during imaging. Included embryos were soaked in 0.016% tricaine solution to prevent the gel from drying out. Embryos were acquired for 6 h every 5 min (magnification 20X, step size 0.5μm). Images were processed and analyzed using the NIS Element Analysis Software (Nikon) and the Fiji (ImageJ) software.

The intracellular level of ROS is an important biomarker for oxidative stress, and an increased ROS level generally indicates a threat to genomic integrity. The production of ROS was estimated with the fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA). H2DCF is non-fluorescent, but in the presence of intracellular ROS it is oxidized to highly fluorescent dichlorofluorescein (DCF). The intracellular ROS level in the control and treated PC12 was analyzed by using the dichlorodihydrofluorescein diacetate (DCFDA) assay (Thermo Fisher Italia, Monza, Italy), according to the manufacturer’s instructions. The ROS derivatives were quantified on a Guava easyCyte flow cytometer (Merck Millipore, Burlington, MA, USA), following the manufacturer’s instructions. These stained cells were spread on a glass slide and stained with DAPI. Next, these slides were observed with a fluorescent microscope (Nikon Eclipse Ci) (Ex-465–495 nm) under low light conditions to reduce photo-bleaching. The image analysis and processing were carried out using NIS-Element analysis software (Nikon, Milan, Italia).