Overview
This video describes a plate reader-based assay to measure mitochondrial calcium uptake in isolated mitochondria. The assay analyzes the fluorescence signal of a calcium-sensitive fluorescent dye to study the kinetics of mitochondrial calcium uptake and calcium overload.
Protocol
1. Reagents and Solutions
- 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.
- 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.
- Prepare 1 mM calcium green-5N stock in dimethyl sulfoxide (DMSO). The calcium green-5N stock can be aliquotted and stored at -20 °C.
- Prepare substrates for Ca2+ uptake.
- Prepare 1 M sodium pyruvate, pH 7.4. Store it in aliquots at -20 °C.
- Prepare 500 mM malate, pH 7.4. Store it in aliquots at -20 °C.
2. Isolation of Cardiac Mitochondria
- 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. - 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. - 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. - Mince the heart into small pieces in 5 mL of ice-cold 1x PBS using a pair of sharp scissors.
- 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.
- 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. - Transfer the homogenate to a 15 mL tube.
- Centrifuge the homogenate at 600 x g at 4 °C for 5 min to pellet nuclei and unbroken cells.
- 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.
- Discard the supernatant and keep the mitochondrial pellet on ice.
- 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.
- 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. - 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.
- 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. - 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. - Add 200 μg of mitochondria to an individual well of a 96-well plate.
- Add the appropriate volume of KCl buffer to the well such that the total volume of mitochondria and KCl buffer comes to 197 μL.
- 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.
- 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. - Start the pre-programmed kinetic protocol and monitor calcium green-5N fluorescence.
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Materials
Name | Company | Catalog Number | Comments |
Synergy Neo2 Multimode microplate reader with injectors | Biotek | ||
Tissue Homogenizer | Kimble | 886000-0022 | |
96-well plate | Corning | 3628 | |
Calcium Green-5N | Invitrogen | C3737 | |
DMSO | Invitrogen | D12345 | |
HEPES | Sigma | H3375 | |
EGTA | Sigma | E8145 | |
Potassium chloride | Fisher | BP366-500 | |
Magnesium chloride | Sigma | M2670 | |
Sodium pyruvate | Sigma | P2256 | |
L-malic acid | Sigma | M1125 | |
Calcium chloride | Sigma | C4901 | |
Potassium phosphate monobasic | Sigma | P0662 |