This method helps to observe mitophagy in living cells using a cell permeant green-fluorescent mitochondria dye and a red-fluorescent lysosome dye. Mitophagy plays an important role in maintaining mitochondria homeostasis and various aspects of cellular function. This technique could provide a new approach to the treatment of mitochondria-related diseases.
This procedure is not very complicated. Capturing clear images is very important for counting the overlay of mitochondria and lysosomes. To begin, culture mouse embryonic fibroblast or MEF cells in a 10 centimeter cell culture dish with 10 milliliters of Dulbecco's Modified Eagle Medium or DMEM.
Incubate at 37 degrees Celsius and 5%carbon dioxide and monitor the cells under a microscope at 100X magnification. When the cells reach 80 to 90%confluency, wash them with two milliliters of Dulbecco's phosphate buffered saline. Then add two milliliters of 0.05%trypsin EDTA for one minute to dissociate the cells, followed by two milliliters of DMEM to stop the reaction.
Centrifuge the cell suspension at 100 G for three minutes and resuspend the pellet in one milliliter DMEM. Count the cells using an automated cell counter and cell counting chamber slides and inoculate 1.5 times 10 to the sixth cells into a new 10 centimeter cell culture dish containing 10 milliliters of DMEM. For the mitophagy assay, prepare a cell suspension as described and dilute the cell suspension to one times 10 to the fifth cells per milliliter of fresh DMEM.
Add two milliliters of the diluted cell suspension to a 20 millimeter confocal dish and shake the dish in a cross. Incubate at 37 degrees Celsius and 5%carbon dioxide for 24 hours. Prepare working solutions of the dyes by diluting the stock solutions.
Add two microliters each of 0.2 millimolar mitochondrial dye and 50 micromolar lysosome dye to two milliliters of DMEM to obtain a working concentration of 0.2 micromolar mitochondrial dye and 50 nanomolar lysosome dye. Remove the medium from the confocal culture dish and add one milliliter of the staining solution to cover the cells. Place the cell culture dish at 37 degrees Celsius and 5%carbon dioxide for 20 to 30 minutes.
Set the parameters of the confocal microscopy imaging software. For dual excitation images, use sequential excitation at 488 nanometers and 543 nanometers and collect emission at 505 to 545 nanometers and greater than 560 nanometers respectively. Remove the culture medium dish containing the dye from the incubator and add one milliliter of Krebs-Henseleit or KH buffer to the dish.
To induce mitophagy, treat the cells with FCCP at one micromolar in KH buffer for 10 minutes at room temperature and immediately proceed to image the cells using the confocal microscope. Apply an appropriate amount of oil to the top of the 63X oil lens. Place the sample on the sample stage of the confocal microscope and move it directly above the objective lens.
Use the imaging software to find the sample by clicking on the Locate tab in the top left corner of the software interface. Select a green filter set for the experiment. Use the coarse adjustment knob to quickly focus by moving the objective lens up and down.
After the cell sample is clearly visible through the eyepiece, search and focus the area of single cells and move it to the center of the field of view. Click on the Acquisition tab in the top left corner of the software interface to acquire images. Select only the 488 nanometer channel and the frame resolution 1024 by 1024 for preview.
Click on the Live tab in the top left corner to start a live scan. Adjust the field of view to the sharpest and adjust the laser power by moving the slider left or right. Keep the gain setting below 600 to avoid over exposure.
Adjust the pinhole value to 156, gain value to 545 and digital offset value to zero. Select the best field of view, check the two channels and choose the frame resolution 1024 by 1024. Click Snap to acquire 2D images and save the acquired images.
In this study, FFCP was used to trigger mitophagy in MEF cells for confocal imaging. Representative images of cells stained with the green-fluorescent mitochondria dye showing mitochondria and stained with the red-fluorescent lysosome dye showing lysosome are shown here. When damaged green-stained mitochondria are engulfed by red-stained lysosomes, the green and red fluorescence overlap to reveal yellow co-localized mitochondria lysosomes.
The yellow dots correspond to these co-localized mitochondria lysosomes representing ongoing mitophagy and thus can be counted to evaluate the extent of mitophagy. Preparation of a working solution of dyes for mitochondria and lysosome staining and maintaining appropriate cell confluency when imaging with confocal microscopy are key steps to remember. This procedure can also be used to analyze mitochondrial number and morphology which is important for assessing mitochondrial dynamics.
Mitophagy is closely associated with a variety of human diseases and this technique can be used to explore the connection between mitophagy and human diseases.
