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.