Degradation of damaged mitochondria by mitophagy is essential for the function of the mitochondrial network. Unfortunately, this process declines with age. We develop and use drugs that activate mitophagy, thereby promoting healthy aging to understand the function of mitophagy in aging and age-related diseases, including Alzheimer's.
One of the biggest challenges is to measure the mitophagy process without affecting it. Recent studies have shown that GFP expression in the mitochondria of muscle cells induces stress that is empty UPR. Therefore, using dyes in non-transgenic animals or cells is essential and compliments genetic technologies.
Our protocol has three main advantages. First, there is no need to generate transgenic animals or cells expressing mitophagy biosensors. Second, no need for expensive infrastructure and expertise such as in case of electron microscopy.
And third, the organelle dioctyl is commercially available and the protocol is simple and fast. The new mitophagy activating compound VL850 induces robust mitophagy and in this way protects from oxidative stress and promote lifespan. Now the big question is to determine the underlying mechanism of action.
We explore the mechanism at multiple levels, from the cellular to the organismal, using C.elegans and mammalian cells. To begin, take the synchronized young adult C.elegans hermaphrodites in a micro centrifuge tube. Add 200 microliters of M9 buffer containing poloxamer 188, pluronic F127, and two microliters of the staining kit reagent to the worm pellet.
Cover the tubes with aluminum foil to protect the dyes from light. Let the mixture rotate at 20 RPM for one hour at room temperature. Then spin the worms at 500 G for one minute.
After that, remove the staining solution without disturbing the worm pellet. Wash the worms with M9 buffer thrice and transfer them into a seeded NGM agar plate containing the appropriate treatment. Wash the worms off the plates into fresh micro centrifuge tubes with one milliliter of M9 buffer.
Afterward, fix the worms with 1%formaldehyde on ice for 30 minutes and wash the worms with one milliliter of M9 buffer thrice to remove any residual formaldehyde. After pelleting the worms, aspirate the maximum amount of supernatant, keeping the pellet intact in 10 microliters of M9.Next, to prepare 2.5%agarose, weigh 0.125 grams of agarose into a 10 milliliter borosilicate glass test tube. Add five milliliters of M9 buffer and dissolve the agarose by gently heating the tube with a bunsen burner.
Transfer the melted agarose to a dry bath set at 75 degrees Celsius. Add 100 microliters of the melted agarose onto a deck glosser microscope cover glass using a one milliliter tip. Immediately put another slide perpendicularly on the agarose drop, forming a cross shape.
After two minutes, gently separate the slides by pushing the upper cover glass, leaving the agarose pad on the bottom cover slide. Transfer the worms onto the agarose pad with a Pasteur glass pipette. Use a wick made of a laboratory wipe to remove excess liquid.
Then cover the worms with a smaller cover slip and apply transparent nail polish to the periphery to prevent evaporation. Place the slide in a dark box to protect it from light. Use a confocal microscope to image the worms within 24 hours at the appropriate wavelengths using a 60 times magnification lens.
Next, open the imaging software and right click on the gray area. From the popup that appears, select Acquisition, TI2 full pad, ND acquisition and look up tables. Under TI2 full pad, select 60 times and adjust the microscope fine focus knob to bring the worms into focus.
Then select spinning disc and choose the 16 bit no-binning option. For each fluorescence filter, set the exposure time to 500 milliseconds and 20 milliseconds for brightfield. Once these parameters are set, select run now and wait for the output image to be generated as an ND2 file.
After imaging the drug treated C.elegans, open the ND file in the Image J server with the colocalization plugin and select the split images option in the dialogue box. Each ND file contains image planes taken at three wavelengths and visible light. Work with brightfield, 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 D.Then reduce the background by generating another image duplicate as demonstrated earlier. Subtract the background with a rolling of 100 and select the create background option to generate an image with the given images background. Now, go to process, click Image Calculator and subtract the first duplicated image from the second duplicated image.
Use 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 is generated as output. Next, focus on the puncta in the head body wall muscle of the worms by manually selecting the area and creating a mask by clicking Edit, Selection, and Create Mask to Select the Region of Interest.
To select the particles in the region of interest, use the image calculator. To select the colocalized 8 bit and mask images, utilize the operation, and to select the puncta in the region of interest, generating an image with puncta in the region of interest. Finally, to analyze the area of the co localized mitochondria and lysosomes, select Analyze followed by Analyze Particles and measure the summation of the puncta between 0.1625 square micrometers and four square micrometers.
To begin, take 70 to 80%confluent Hep3B cells. Remove 250 microliters of medium from each of the wells and add 50 microliters of medium with the appropriate treatment or vehicle. Add 50 microliters of medium to each well containing the staining reagent and incubate the cells with the dye.
After removing the medium and washing the cells with pre-warmed PBS, fix the cells with 200 microliters of fixing solution for 15 minutes at room temperature. Afterward, decant the fixative solution and wash briefly with 200 microliters of PBS. Finally, add 200 microliters of PBS.
Keep the cells covered and protected from light at four degrees celsius and image within 24 hours. After imaging the drug treated Hep3B 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 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 eight 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 two 50.0. An 8 bit binary image containing co localized 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 points 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 co localized 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 Hep3B cells displayed colocalization of mitochondria and lysosomes. VL850 and FCCP induced significant mitophagy in the Hep3B cells.
