mitochondrial calcium uptake assay: a plate reader-based method for measuring calcium uptake in isolated mitochondria

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

To start mitochondrial calcium uptake measurement using a plate reader, program the reader to perform a kinetic read of calcium green-5N fluorescence every second, for a total time of 1,000 seconds.
Program the reagent injectors to dispense 5 microliters of calcium chloride solution at various time points. Then, prime the injectors with the calcium chloride solution that will be used.
Next, add 200 micrograms of isolated mitochondria to one well of a 96-well plate, and add the appropriate volume of potassium chloride buffer to achieve a total volume of 197 microliters. Then, add 1 microliter of 1 molar pyruvate, 1 microliter of 500 millimolar malate, and 1 microliter of 1 millimolar calcium green-5N stock, and mix gently by pipetting.
Incubate protected from light at room temperature for 2 minutes, for mitochondria to become energized. Finally, start the pre-programmed kinetic protocol, and monitor calcium green-5N fluorescence.

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1. Reagents and Solutions
<ol>
	<li>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(&#946;-aminoethyl ether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA), pH adjusted to 7.4 with KOH. Sterilize it through a 0.22 &#956;m filter and store it at 4 &#176;C. Ensure that the MS-EGTA buffer is pre-chilled to 4 &#176;C before use.</li>
	<li>Prepare 100 mL of KCl Buffer: 125 mM KCl, 20 mM HEPES, 1 mM KH2PO4, 2 mM MgCl2, 40 &#956;M EGTA and pH adjusted to 7.2 with KOH. Store it at room temperature.</li>
	<li>Prepare 1 mM calcium green-5N stock in dimethyl sulfoxide (DMSO). The calcium green-5N stock can be aliquotted and stored at -20 &#176;C.</li>
	<li>Prepare substrates for Ca2+ uptake.
	<ol>
		<li>Prepare 1 M sodium pyruvate, pH 7.4. Store it in aliquots at -20 &#176;C.</li>
		<li>Prepare 500 mM malate, pH 7.4. Store it in aliquots at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Isolation of Cardiac Mitochondria
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Mince the heart into small pieces in 5 mL of ice-cold 1x PBS using a pair of sharp scissors.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Transfer the homogenate to a 15 mL tube.</li>
	<li>Centrifuge the homogenate at 600 x g at 4 &#176;C for 5 min to pellet nuclei and unbroken cells.</li>
	<li>Transfer the supernatant to a fresh 15 mL tube and centrifuge it at 10,000 x g at 4 &#176;C for 10 min to pellet mitochondria.</li>
	<li>Discard the supernatant and keep the mitochondrial pellet on ice.</li>
	<li>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 &#176;C for 10 min, and discard the supernatant.</li>
	<li>After the final wash, discard the supernatant and resuspend the mitochondria in 100 &#956;L of ice-cold MS-EGTA buffer. Keep the mitochondria on ice. 
	NOTE: Mitochondria should be used for experimentation within 1 h.</li>
	<li>Measure mitochondrial protein concentration using a Bradford Protein Assay.</li>
</ol>
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.
<ol>
	<li>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 &#956;L 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.</li>
	<li>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.</li>
	<li>Add 200 &#956;g of mitochondria to an individual well of a 96-well plate.</li>
	<li>Add the appropriate volume of KCl buffer to the well such that the total volume of mitochondria and KCl buffer comes to 197 &#956;L.</li>
	<li>Add 1 &#956;L of 1 M pyruvate and 1 &#956;L 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.</li>
	<li>Add 1 &#956;L 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.</li>
	<li>Start the pre-programmed kinetic protocol and monitor calcium green-5N fluorescence.</li>
</ol>

<table><tbody><tr><td>Synergy Neo2 Multimode microplate reader with injectors</td><td>Biotek</td><td></td><td></td></tr><tr><td>Tissue Homogenizer</td><td>Kimble</td><td>886000-0022</td><td></td></tr><tr><td>96-well plate</td><td>Corning</td><td>3628</td><td></td></tr><tr><td>Calcium Green-5N</td><td>Invitrogen</td><td>C3737</td><td></td></tr><tr><td>DMSO</td><td>Invitrogen</td><td>D12345</td><td></td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375</td><td></td></tr><tr><td>EGTA</td><td>Sigma</td><td>E8145</td><td></td></tr><tr><td>Potassium chloride</td><td>Fisher</td><td>BP366-500</td><td></td></tr><tr><td>Magnesium chloride</td><td>Sigma</td><td>M2670</td><td></td></tr><tr><td>Sodium pyruvate</td><td>Sigma</td><td>P2256</td><td></td></tr><tr><td>L-malic acid</td><td>Sigma</td><td>M1125</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma</td><td>C4901</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma</td><td>P0662</td><td></td></tr></tbody></table>

Source: Maxwell, J.T., et al., <a href="https://app.jove.com/v/57225">Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells.</a> J. Vis. Exp. (2018).
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.

Source: Maxwell, J.T., et al., Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells. J. Vis. Exp. (2018).
This video ...

Mitochondria take up cytosolic calcium as it is essential for their proper functioning.
Excessive calcium influx into the mitochondrial matrix - mitochondrial calcium overload - triggers the opening of an inner membrane channel, the mitochondrial permeability transition pore, or MPTP. The opened pore allows increased calcium efflux from the mitochondria, causing mitochondrial dysfunction.
To measure the mitochondrial calcium uptake, begin with a multi-well plate containing isolated mitochondria suspended in a suitable buffer. Supplement the well with pyruvate and malate, which are ATP-producing substrates. Mix the contents of the well, and incubate.
During incubation, pyruvate and malate are converted to ATPs, energizing the mitochondria. Add a solution containing mitochondrial membrane-impermeable, calcium-sensitive fluorescent dye molecules. Dispense the calcium-containing buffer to the energized mitochondria.
Monitor the dye&#39;s calcium fluorescence as the dye molecules bind to the free calcium in the buffer.
Add more calcium ions to initiate mitochondrial calcium influx. The gradually increasing mitochondrial calcium decreases the calcium ions in the buffer, reducing the dye&#39;s calcium fluorescence.
Once the mitochondria reach their maximum calcium-carrying capacity, the further addition of calcium ions open the MPTP channels and import calcium into the buffer. The buffer calcium binds to the dye molecules, causing increased dye calcium fluorescence.
Plot the dye fluorescence at different time points of calcium addition to monitor the kinetics of mitochondrial calcium uptake.

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

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