Mitochondrial Calcium Uptake Assay: A Plate Reader-Based Method for Measuring Calcium Uptake in Isolated Mitochondria

1. Reagents and Solutions

1. Make 500 mL of MS-EGTA buffer for mitochondrial isolation: 225 mM mannitol, 75 mM sucrose, 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), pH adjusted to 7.4 with KOH. Sterilize it through a 0.22 μm filter and store it at 4 °C. Ensure that the MS-EGTA buffer is pre-chilled to 4 °C before use.
2. Prepare 100 mL of KCl Buffer: 125 mM KCl, 20 mM HEPES, 1 mM KH₂PO₄, 2 mM MgCl₂, 40 μM EGTA and pH adjusted to 7.2 with KOH. Store it at room temperature.
3. Prepare 1 mM calcium green-5N stock in dimethyl sulfoxide (DMSO). The calcium green-5N stock can be aliquotted and stored at -20 °C.
4. Prepare substrates for Ca²⁺ uptake.
   1. Prepare 1 M sodium pyruvate, pH 7.4. Store it in aliquots at -20 °C.
   2. Prepare 500 mM malate, pH 7.4. Store it in aliquots at -20 °C.

2. Isolation of Cardiac Mitochondria

1. Euthanize the mouse according to institutional standards.
   NOTE: For our institutionally approved method of euthanasia, mice were anesthetized by isofluorane inhalation and sacrificed by cervical dislocation.
2. Collect the heart by opening the chest cavity, cutting along either side of the ribs, flanking the heart, cutting away the diaphragm, and excising the heart tissue.
   NOTE: In this protocol, mitochondrial isolation is performed from a whole heart collected from an adult mouse (approximately 120 mg), which should be sufficient for 3-4 experiments. For mitochondrial isolation from other mitochondria-rich tissues such as the liver, up to 200 mg of tissue can be used following the protocol described.
3. Rinse the tissue thoroughly in 25 mL of ice-cold 1x phosphate-buffered saline (PBS) ensuring that all blood is squeezed out of the ventricles.
   NOTE: The heart will be sufficiently rinsed when the liquid squeezed out of the heart runs clear.
4. Mince the heart into small pieces in 5 mL of ice-cold 1x PBS using a pair of sharp scissors.
5. Discard the PBS and transfer minced heart tissue to a pre-chilled 7 mL glass-Teflon Dounce homogenizer with a 0.10 to 0.15 mm clearance.
6. Add 5 mL of ice-cold MS-EGTA buffer, and homogenize the sample until tissue pieces are no longer visible (approximately 11 strokes).
   NOTE: Do not over-homogenize the tissue as the goal is to obtain intact and functional mitochondria.
7. Transfer the homogenate to a 15 mL tube.
8. Centrifuge the homogenate at 600 x g at 4 °C for 5 min to pellet nuclei and unbroken cells.
9. Transfer the supernatant to a fresh 15 mL tube and centrifuge it at 10,000 x g at 4 °C for 10 min to pellet mitochondria.
10. Discard the supernatant and keep the mitochondrial pellet on ice.
11. Wash the mitochondrial pellet twice using ice-cold MS-EGTA buffer. For each wash, resuspend the mitochondrial pellet in 5 mL of MS-EGTA buffer, centrifuge it at 10,000 x g at 4 °C for 10 min, and discard the supernatant.
12. After the final wash, discard the supernatant and resuspend the mitochondria in 100 μL of ice-cold MS-EGTA buffer. Keep the mitochondria on ice.
    NOTE: Mitochondria should be used for experimentation within 1 h.
13. Measure mitochondrial protein concentration using a Bradford Protein Assay.

3. Plate Reader-based Measurement of Mitochondrial Calcium Uptake

NOTE: Here, it is described the protocol for analyzing mitochondrial Ca²⁺ uptake using a multimode plate reader fitted with injectors. Any plate reader with the capability of reading calcium green-5N fluorescence (excitation/emission of 506/532 nm) in a kinetic mode with automated reagent injectors to keep the reaction protected from light can be used.

1. Program the plate reader to perform a kinetic read of calcium green-5N fluorescence with measurements taken every second for a total assay time of 1,000 s. Additionally, program the reagent injectors to dispense 5 μL of CaCl₂ solution at the 30 s, 150 s, 300 s, 480 s, and 690 s time points.
   NOTE: The timing of the CaCl₂ injections is user-defined, and can be adjusted according to experimental needs.
2. Prime the reagent injectors with the CaCl₂ solution to be used.
   NOTE: The concentration of the CaCl₂ solution is user-defined, and can be adjusted in subsequent runs to titrate the amount of Ca²⁺ required to trigger MPTP opening.
3. Add 200 μg of mitochondria to an individual well of a 96-well plate.
4. Add the appropriate volume of KCl buffer to the well such that the total volume of mitochondria and KCl buffer comes to 197 μL.
5. Add 1 μL of 1 M pyruvate and 1 μL of 500 mM malate to the mitochondria mixture. Pipet gently to mix, and incubate the mitochondria with the substrates for 2 min at room temperature to allow mitochondria to become energized.
6. Add 1 μL of 1 mM calcium green-5N stock. Mix gently by pipetting.
   NOTE: Protect the reaction from light following the addition of the calcium green-5N dye.
7. Start the pre-programmed kinetic protocol and monitor calcium green-5N fluorescence.