This protocol provides a reference for the technique approach to the study of autophage and it describes the entire experimental procedures in detail. The main advantage of this technique is that it allows the length observation of the change in autophagic flux. Before starting the experiment, determine the effective infection concentration and taking necessary safety measures.
Begin by centrifuging neutralized H9C2 myocardial cells at 179G for five minutes at room temperature. Discard the supernatant and mix the cells in DMEM complete medium by gently blowing. Divide the cells into nine equal proportions.
Add dimethyl sulfoxide to the control group. Then, add varying concentrations of crocetin to the remaining groups. Seed 100 microliters of cells per well in a 96-well plate.
Once incubated for 24 hours, discard the supernatant. Then, wash the cells three times with PBS. Add 100 microliters of DMEM basal medium to each well and incubate the cells for four hours at 37 degrees Celsius.
Now, add 20 microliters of MTS and incubate for another two hours as demonstrated. Finally, measure the absorbance at 490 nanometers and calculate the cell viability. To determine ROS, dilute DCFH-DA at a 1:1000 ratio with the serum-free medium.
Discard the cell's supernatant from a 48-well plate, previously seeded with H9C2 cells. Then, wash the cells twice with a serum-free medium. Add 150 microliters of diluted DCFH-DA to each well and incubate it at 37 degrees Celsius for 20 minutes.
Next, wash the cells three times with serum-free medium. Finally, capture images using a fluorescence microscope. To analyze mitochondrial membrane potential, add JC-1 working solution to the previously cultured cells and mix well.
Incubate them at 37 degrees Celsius for 20 minutes. Then, wash the cells twice with JC-1 staining buffer. Add complete medium to each well and capture photographs using a fluorescence microscope.
Fix the cells with cell fixation solution for 30 minutes at room temperature. Then, wash them with PBS once. Add 0.3%Triton-100 to each well and incubate at room temperature for five minutes.
After two PBS washes, add TUNEL detection working solution to each well and incubate the cells at 37 degrees Celsius in the dark for 60 minutes. Add DAPI containing an anti-fluorescence quenching tablet and capture the images as demonstrated. Replace half of the medium from the previously cultured cells with fresh medium in each well of a 48-well plate.
Add the mCherry GFP-LC3B adenovirus followed by polybrene to improve the infection efficiency. Replace the medium every 24 hours. Simultaneously using a confocal microscope, observe fluorescent protein expression to confirm infection.
Once infected, culture and capture the cells as described in the manuscript. Begin by fixing previously cultured cells with 4%paraformaldehyde for 10 minutes at room temperature and add 0.1%Triton-100. Next, block the cells with an animal-free block solution for one hour.
Then, incubate them with a primary antibody at four degrees Celsius overnight. The next day, add a fluorescent secondary antibody to the cells and incubate them in the dark for one hour. Finally, add the DAPI containing anti-fluorescence quenching tablets before capturing the images.
In the present study, crocetin had a significant proliferative effect on cells. While a concentration above 200 micromolar inhibited the proliferation of H9C2 cells. The cell viability was reduced considerably after hydrogen peroxide treatment.
However, crocetin reversed this change to a certain extent. The enhanced ROS level after hydrogen peroxide treatment was reversed by crocetin to some extent. The fluorescence results also confirmed reduced ROS with crocetin.
Further, crocetin addition restored the mitochondrial membrane potential collapses caused by hydrogen peroxide treatment. Similarly, apoptosis was also reversed by crocetin. In addition, the crocetin pre-treatment reversed the autophagy flow of H9C2 cardiomyocytes, induced due to hydrogen peroxide treatment.
Crocetin pre-treatment also reduced the translocation of Parkin mitochondria induced by hydrogen peroxide treatment. Following this procedure, electron microscopic can also be used to directly observe a microphage, allowing for a more intuitive view of mitochondria being engulfed by autophagosomes.
