After imaging the drug treated Hep-3B cells, open the confocal images in image J with the colocalization plugin. Open the ND file in the image J server and select the split channels option in the dialogue box. Work with green and red images.
Generate duplicates of these images to keep the original image untouched by clicking on image and selecting duplicate or using the keyboard shortcut shift plus D.To reduce the background, generate another image duplicate, subtract the background with a rolling radius of 100, and select the create background option to generate an image with the given images background. Then, go to process. Click image calculator and subtract the first duplicated image from the second duplicated image.
Utilize the resulting images for the colocalization analysis. To use the colocalization plugin, convert the green and red channel images to 8 bit by clicking image, selecting type, followed by 8 bit. Next, click on plugins and select colocalization.
To measure the colocalization of mitochondria and lysosome signals, set the ratio to 75%the threshold red channel to 80.0, and the threshold green channel to 50.0. An 8 bit binary image containing colocalized puncta and combining the three 8 bit images in an RGB image will be generated. Convert these images to 8 bit images.
To obtain the region of interest of the cells, generate an image that highlights the area of cells by selecting a colocalized point's image. To select the entire cell area, select the resultant colocalized points image and click on image, adjust threshold, and set threshold to ensure all the cell area is highlighted. To analyze the area of the cell, select analyze, click analyze particles, and measure all the particles in the image from zero to infinity, the default setting for analyze particles.
To analyze the area of the colocalized mitochondria and lysosomes, select the colocalized 8 bit image. Then, select analyze. Click analyze particles and measure the summation of puncta between 0.1625 square micrometers and four square micrometers.
Divide the area of the colocalized mitochondria and lysosomes by the total cell area to measure the colocalized puncta. The confocal images of Hep-3B cells displayed colocalization of mitochondria and lysosomes. VL 850 and FCCP induced significant mitophagy in the Hep-3B cells.
