Name: mitochondrial ca2+ retention capacity assay and ca2+-triggered mitochondrial swelling assay
Uploaded: 2018-05-01T12:30:00
Description: Watch this Scientific Journal Video about Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay at JoVE.com

This study was supported by grants from grant of the Outstanding Scientist of Shandong (JQ201421) and grant from NSFC (81371226).

1. Isolation of Mitochondria
NOTE: All solutions and equipment should be precooled to 0 - 4 &#176;C and kept on ice.
<ol>
	<li>Seed about 3 x 106 SH-SY5Y cells per 10 cm cell culture dish. Culture cells in high-glucose Dulbecco&#39;s modified Eagle medium with 10% fetal bovine serum, penicillin (100 U/mL)-streptomycin (100 &#181;g/mL). Maintain at 37 &#176;C in an incubator containing 5% CO2 overnight. 
	NOTE: At least 1 - 2 x 107 SH-SY5Y cells are neede...

<ol>
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Here, we described a simple and effective protocol for the mitochondrial Ca2+ retention capacity assay and Ca2+-triggered mitochondrial swelling assay.
For the mitochondrial isolation, make sure that all the materials and tubes are on ice, especially when homogenizing the cells. 0.25% trypsin-EDTA can also be used to detach cells from the dish for 5 min at 37 &#176;C. After 15 - 25 strokes, it is necessary to observe the intact cell membrane under the microscope and avoid...

Mitochondria are the main cellular organs to produce nearly 95% of the ATP used in the mammalian cells by oxidative phosphorylation. It is reported that the sequestered micromolar concentration of Ca2+ by mitochondria, the presence of ADP, and inorganic phosphate can be used to phosphorylate ADP to synthesis ATP1. When the concentration of cytosolic Ca2+ goes above a threshold, mitochondria can uptake Ca2+ rapidly and efflux it slowly. Thus, the functioning mitochondria can influx the increased cytosolic Ca2+. Irrelevant to the participation in the oxidative phosphorylation, the mitochondrial Ca2+ also take part in the cytosolic calcium signals and activate the mitochondrial apoptotic mechanism by inducing the opening of the mPTP in the inner mitochondrial membrane2. It has been widely recognized that abnormal elevation of intracellular Ca2+ can induce mitochondrial massive swelling by opening the mPTP3. Thus, this protocol aims to assess the mitochondrial function by quantifying the mitochondrial Ca2+ retention capacity and Ca2+-trigged mitochondrial swelling.
Ca2+ retention capacity is the measurement of the ability of mitochondria to uptake cytosolic calcium. Mitochondria can buffer the cytosolic free calcium and regulate calcium-dependent cellular processes by calcium uptake in the form of inactive precipitates. Impaired Ca2+ retention capacity occurs in stress phenomena associated with energy limitation and even neurodegenerative diseases4,5. Isolated mitochondria or digitonin-permeabilized cells can be used to identify the Ca2+ retention capacity, and the elevated ability to accumulate Ca2+ by isolated mitochondria with the additonal glutamate and malate is not effected by the mitochondrial isolation procedure6. A titrated amount of digitonin should be used to permeabilize the plasma membranes of different cell types. The hexapotassium salt of Ca2+-binding green fluorescent dye, a Ca2+-sensitive cell-impermeant visible light-excitable indicator, has been widely used7. Ca2+-binding green fluorescent dye is a low-affinity indicator and used to show that intracellular free Ca2+ concentrations continue to rise during prolonged (5 min) stimulations8. This method was less sensitive than the radioactive filter technique but was greatly simplified. The additional Ca2+ elevates calcium green fluorescence and the Ca2+ uptake by mitochondria returns the fluorescence to baseline. Sequential additions of Ca2+ were made until the mitochondria failed to uptake extramitochondrial Ca2+ 9. A fluorescence microplate reader can be used to continuously report the calcium green fluorescence.
After Ca2+ accumulation, mitochondria depolarized, released Ca2+ into the medium, and began to swell. Ca2+-triggered mitochondria swelling is used to indicate the mPTP opening. Electron microscopy and the decrease in light absorbance at 540 nm can be used to measure the Ca2+-triggered mitochondrial swelling10,11. Mitochondria volume can be directly determined by forward angle light scattering12, where decreases in the absorbance reflect passive swelling of the mitochondrial matrix.
Here, we illustrate the methodology to examine the mitochondrial Ca2+ retention capacity and Ca2+-triggered mitochondrial swelling in isolated mitochondria from SH-SY5Y cells.

<table>
	<tbody>
		<tr>
			<td>SH-SY5Y cell line</td>
			<td>ATCC (The Global Bioresource Center)</td>
			<td>ATCC Number: CRL-2266</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium green-5N</td>
			<td>Life Technologies</td>
			<td>c-3737</td>
			<td>Ca2+-binding green fluorescent dye</td>
		</tr>
		<tr>
			<td>complete protease inhibitors</td>
			<td>Roche Molecular Biochemicals</td>
			<td>4693116001</td>
			<td></td>
		</tr>
		<tr>
			<td>glass homogenizer</td>
			<td>Kimble Chase</td>
			<td>9885303002</td>
			<td></td>
		</tr>
		<tr>
			<td>glutamate</td>
			<td>Sigma-Aldrich</td>
			<td>RES5063G-A7</td>
			<td></td>
		</tr>
		<tr>
			<td>malate</td>
			<td>Sigma-Aldrich</td>
			<td>46940-U</td>
			<td></td>
		</tr>
		<tr>
			<td>rotenone</td>
			<td>Sigma-Aldrich</td>
			<td>R8875</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>00457</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Sigma-Aldrich</td>
			<td>V900050</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>V900266</td>
			<td></td>
		</tr>
		<tr>
			<td>Varioskan flash instruments</td>
			<td>Thermo Scientific</td>
			<td>IC100E</td>
			<td></td>
		</tr>
		<tr>
			<td>Bongkrekate</td>
			<td>Biovision</td>
			<td>1820-100</td>
			<td></td>
		</tr>
		<tr>
			<td>atractyloside</td>
			<td>Sigma-Aldrich</td>
			<td>C4992</td>
			<td></td>
		</tr>
		<tr>
			<td>high-glucose Dulbecco&#8217;s modified Eagle medium</td>
			<td>Hyclone</td>
			<td>SH30022</td>
			<td>4.5 g/L glucose, L-glutamine, without sodium pyruvate</td>
		</tr>
		<tr>
			<td>fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SV30087</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin</td>
			<td>Sigma-Aldrich</td>
			<td>P3032</td>
			<td></td>
		</tr>
		<tr>
			<td>streptomycin</td>
			<td>Invitrogen</td>
			<td>11860-038</td>
			<td></td>
		</tr>
		<tr>
			<td>BCA assay kit</td>
			<td>Thermo Scientific</td>
			<td>NCI3225CH</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Hyclone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm cell culture dish</td>
			<td>NEST</td>
			<td>704001</td>
			<td></td>
		</tr>
	</tbody>
</table>

mitochondrial ca2+ retention capacity assay and ca2+-triggered mitochondrial swelling assay

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in cytosolic Ca2+ buffering, and the ATP production in mitochondrial can be mediated by intramitochondrial free Ca2+ concentration. Ca2+ retention capacity can be regarded as the capability of mitochondria to retain calcium in the mitochondrial matrix. Accumulated intracellular Ca2+ leads to the permeability of the inner mitochondrial membrane, termed the opening of mitochondrial permeability transition pore (mPTP), which leads to the leakage of molecules with a molecular weight less than 1.5 kDa. Ca2+-triggered mitochondria swelling is used to indicate the mPTP opening. Here, we describe two assays to examine the Ca2+ retention capacity and Ca2+-triggered mitochondrial swelling in isolated mitochondria. After certain amounts of Ca2+ are added, all steps can be completed in one day and recorded by a microplate reader. Thus, these two simple and effective assays can be adopted to assess the Ca2+-related mitochondrial functions.

Representative Results of Ca2+ Retention Capacity: 
Results were expressed as fluorescence values. With the additional pulses of Ca2+ (200 nmols/mg mitochondrial protein), the fluorescence increased by double above baseline with the mPTP opening. 5 &#956;M Bongkrekate (BKA), an inhibitor of Ca2+-induced mPTP opening, or 1 &#956;M atractyloside (ATR), an activator of Ca2+-induced mPTP opening, were added in isolated mitochondria. Th...

Watch this Scientific Journal Video about Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay at JoVE.com

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

This protocol aims to describe a method to examine the Ca2+ retention capacity and Ca2+- triggered mitochondrial swelling of isolated mitochondria of SH-SY5Y cells step-by-step.

Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay

The production of ATP by oxidative phosphorylation is the primary function of mitochondria. Mitochondria in higher eukaryotes also participate in ...

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