Determination of the Calcium Retention Capacity of Isolated Mitochondria

Place the mitochondria isolated from neuroblastoma cells in a buffer containing membrane-impermeable, calcium-binding fluorescent dye.

Incubate to allow the mitochondria to stabilize in the buffer.

Add calcium-containing buffer and incubate with shaking.

The calcium ions bind to the dye molecules, causing their fluorescence.

Using a fluorescence spectrometer, monitor the dye's fluorescence in real-time.

Now, inject calcium-containing buffer at regular intervals.

The increased calcium concentration promotes mitochondrial calcium uptake, reducing the free calcium ions available to bind to the dye, which results in decreased fluorescence.

Once the mitochondria reach their maximum calcium storage capacity, further calcium addition triggers the opening of the mitochondrial permeability transition pore (mPTP).

This pore opening releases calcium from the mitochondria into the buffer, where it binds to the dye and causes increased fluorescence.

Determine the total amount of calcium required to induce mPTP opening, which reflects the mitochondrial calcium retention capacity.

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 1 milliliter of isolated mitochondria in potassium chloride media containing 0.5 micromolar calcium-binding green fluorescent dye to each well of a 6-well plate.

Incubate the mitochondria in the calcium-binding green fluorescent dye in the 6-well plate at room temperature. Protect it from ambient light for 1 minute. Following incubation, add 4 microliter aliquots of a 20-millimolar calcium chloride solution to each well of the 6-well plate, using the automatic dispense setting to introduce 200 nanomoles of calcium per milligram of mitochondrial protein.

Use a fluorescent spectrometer to monitor the fluorescence changes every three seconds for 2 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 3 seconds between readings, controlled by software. Add an additional 4-microliter aliquot of 20-millimolar calcium chloride solution to each well, then monitor the fluorescence changes every 3 seconds for 2 minutes.