Mitochondrial Ca²⁺ Retention Capacity Assay and Ca²⁺-triggered Mitochondrial Swelling Assay

The overall goal of this experiment is to determine mitochondrial calcium retention capacity in calcium induced mitochondrial swelling. This method can help answer key questions in the study of mitochondrial dysfunction, such as abnormal energy metabolism. The main advantage of this technique is that it’s very simple and effective.

To begin, seed about three million SH-SY5Y cells per 10 centimeter cell culture dish. Remove the medium and wash the cells with about one milliliter of ice cold PBS in a 10 centimeter cell culture dish three times. Add one milliliter of ice cold PBS into the 10 centimeter cell culture dish and scrape the adherent cells into a new 1.5 milliliter tube.

Centrifuge at 800 times G for 10 minutes at four degrees Celsius. During the centrifugation, prepare five milliliters of mitochondrial isolation buffer, supplemented with 50 microliters of protease inhibitor stock solution. Remove the supernatant and re-suspend the pellet with one milliliter of mitochondrial isolation buffer.

Incubate the 1.5 milliliter tube on ice for 30 minutes. Then, lyse the suspended cells with a glass homogenizer, up and down manually on ice. Transfer the homogenate into a 1.5 milliliter tube.

Then, centrifuge at 1, 000 times G for five minutes at four degrees Celsius to remove the debris. Following centrifugation, transfer the supernatant into a new 1.5 milliliter tube before centrifuging again as before. After transferring the supernatant to a new tube, centrifuge at 14, 000 times G for 15 minutes at four degrees Celsius.

Remove the supernatant and re-suspend the pellet containing mitochondria with one milliliter of mitochondrial isolation buffer. Then centrifuge the solution for 15 minutes at 14, 000 times G and four degrees Celsius to precipitate the mitochondria. The resulting mitochondria pellet must be kept on ice and re-suspended in 50 to 100 microliters of potassium chloride media, according to the pellet volume before any assay.

Finally, use five microliters of mitochondrial solution to measure the mitochondrial protein concentration by standard BCA assay, measuring OD562 using a plate reader. Prepare the potassium chloride media and add the calcium binding green fluorescent dye to a final concentration of 0.5 micromolar before the experiment. To determine mitochondrial calcium retention capacity, first transfer one milliliter of isolated mitochondria in potassium chloride media containing 0.5 micromolar calcium binding green fluorescent dye to each well of a six well plate.

Incubate the mitochondria and the calcium binding green fluorescent dye in the six well plate at room temperature, protected from ambient light for one minute. Following incubation, add four microliter aliquots at the 20 millimolar calcium chloride solution to each well of the six well plate using the automatic dispense setting to introduce 200 nanomoles of calcium per milligram of mitochondrial protein. Use the fluorescent spectrometer to monitor the fluorescence changes every three seconds for two minutes with an excitation wavelength of 506 nanometers and an emission wavelength of 531 nanometers.

The plate is equipped with shaking at 600 rpm for three seconds between readings, controlled by software. Add an additional four microliter aliquot of 20 millimolar calcium chloride solution to each well, then monitor the fluorescence changes every three seconds for two minutes. To determine calcium induced mitochondrial swelling, transfer one milliliter of isolated mitochondria in potassium chloride media to each well of a six well plate.

Then, add 10 microliters of 20 millimolar calcium chloride aqueous solution to the mitochondrial protein to trigger mitochondrial swelling. Using a microplate reader controlled by software, record the decrease in absorbance every three seconds for 10 minutes at 540 nanometers. Representative results of mitochondrial calcium retention capacity of bongkrekic, an inhibitor of the calcium induced mitochondrial permeability transition pore, or MPTP, opening atractyloside, an activator of the calcium induced MPTP opening and a blank control, are shown here.

The capacity on in the bongkrekic treatment is much higher than the atractyloside group, as expected. Representative results of calcium induced mitochondrial swelling of bongkrekic, atractyloside, and the no treatment control are shown here. The decreased absorbance monitored at 540 nanometers indicates the mitochondrial swellings.

The results suggest that BKA can inhibit the MPTP opening, while ATR can facilitate the MPTP opening. Once mastered, this technique can be done in five hours if it is performed properly. While attempting this procedure, it’s important to remember to keep all the solutions on ice.