determination of the calcium retention capacity of isolated mitochondria

Determination of the Calcium Retention Capacity of Isolated Mitochondria

Prepare the potassium chloride media and add the calcium binding green fluorescent dye to a final concentration of 0.5 micromolar before the experiment. To determine mitochondrial calcium retention capacity, first transfer 1 milliliter of isolated mitochondria in potassium chloride media containing 0.5 micromolar calcium-binding green fluorescent dye to each well of a 6-well plate.
Incubate the mitochondria in the calcium-binding green fluorescent dye in the 6-well plate at room temperature. Protect it from ambient light for 1 minute. Following incubation, add 4 microliter aliquots of a 20-millimolar calcium chloride solution to each well of the 6-well plate, using the automatic dispense setting to introduce 200 nanomoles of calcium per milligram of mitochondrial protein.
Use a fluorescent spectrometer to monitor the fluorescence changes every three seconds for 2 minutes, with an excitation wavelength of 506 nanometers and an emission wavelength of 531 nanometers. The plate is equipped with shaking at 600 RPM for 3 seconds between readings, controlled by software. Add an additional 4-microliter aliquot of 20-millimolar calcium chloride solution to each well, then monitor the fluorescence changes every 3 seconds for 2 minutes.

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1. Determination of Mitochondrial Ca2+ Retention Capacity
<ol>
	<li>Prepare the KCl media (125 mM KCl (potassium chloride), 2 mM K2HPO4 (dipotassium phosphate), 1 mM MgCl2 (magnesium chloride), 20 mM HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid), 5 mM glutamate, 5 mM malate, and 2 &#956;M rotenone, pH 7.4) and add the Ca2+-binding green fluorescent dye to a final concentration of 0.5 &#956;M before the experiment.</li>
	<li>Transfer 1 mL of isolated mitochondria (0.4 mg/mL) in KCl media containing 0.5 &#956;M Ca2+-binding green fluorescent dye into each 6-well plate well.</li>
	<li>Incubate the mitochondria and the Ca2+-binding green fluorescent dye in the 6-well plate at room temperature, protected from ambient light for 1 min. Add 4 &#956;L aliquots of a 20 mM CaCl2 (calcium chloride) solution (20 mM CaCl2, 127 mM KCl, 1 mM MgCl2, 20 mM HEPES, 5 mM glutamate, 5 mM malate, pH 7.4) to each 6-well plate well using the automatic dispense setting to introduce 200 nmol Ca2+/mg mitochondrial protein.</li>
	<li>Use a fluorescence spectrometer to monitor the fluorescence changes every 3 s for 2 min with an excitation wavelength of 506 nm and an emission wavelength of 531 nm. The plate is equipped with shaking at 600 rpm for 3 s between readings controlled by software (Figure 1).</li>
	<li>Add an additional 4 &#956;L aliquot of 20 mM CaCl2 solution to each well and monitor the fluorescence changes every 3 s for 2 min. Repeat this step until the fluorescence increases with time. 
	NOTE: Mitochondria are challenged with the added Ca2+ indicated by the increased recording fluorescence. When the mPTP (mitochondrial permeability transition pore) opens, the fluorescence increases with time.</li>
	<li>Interpret the total amount of Ca2+ added until the mPTP opens as the Ca2+ retention capacity.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Calcium green-5N</td>
			<td>Life Technologies</td>
			<td>c-3737</td>
			<td>Calcium binding green fluorescent dye</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>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>457</td>
			<td></td>
		</tr>
		<tr>
			<td>Dipotassium phosphate</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>Calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>V900266</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Li, W., et. al. <a href="https://app.jove.com/v/56236">Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay.</a> J. Vis. Exp. (2018)
This video demonstrates the procedure to assess mitochondrial calcium retention capacity by monitoring real-time changes in fluorescence as mitochondria uptake calcium. This assay is useful for studying mitochondrial function and calcium-related processes, including apoptosis and neurodegenerative diseases.

<img alt="Figure 1" src="/files/ftp_upload/23257/23257_Figure1.jpg" /> 
Figure 1: The software settings for mitochondrial Ca2+ retention capacity assay.

Source: Li, W., et. al. Mitochondrial Ca2+ Retention Capacity Assay and Ca2+-triggered Mitochondrial Swelling Assay. J. Vis. Exp. (2018)
This video ...

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&#39;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.

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비디오: Determination of the Calcium Retention Capacity of Isolated Mitochondria - 실험

Analyses of Mitochondrial Calcium Influx in Isolated Mitochondria and Cultured Cells

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ability to accurately measure the influx and efflux of Ca2+ from mitochondria is important for determining the role of mitochondrial Ca2+ handling in these processes. In this report, we present two methods for the measurement of mitochondrial Ca2+ handling in both isolated mitochondria and cultured cells. We first detail a plate reader-based platform for measuring mitochondrial Ca2+ uptake using the Ca2+ sensitive dye calcium green-5N. The plate reader-based format circumvents the need for specialized equipment, and the calcium green-5N dye is ideally suited for measuring Ca2+ from isolated tissue mitochondria. For our application, we describe the measurement of mitochondrial Ca2+ uptake in mitochondria isolated from mouse heart tissue; however, this procedure can be applied to measure mitochondrial Ca2+ uptake in mitochondria isolated from other tissues such as liver, skeletal muscle, and brain. Secondly, we describe a confocal microscopy-based assay for measurement of mitochondrial Ca2+ in permeabilized cells using the Ca2+ sensitive dye Rhod-2/AM and imaging using 2-dimensional laser-scanning microscopy. This permeabilization protocol eliminates cytosolic dye contamination, allowing for specific recording of changes in mitochondrial Ca2+. Moreover, laser-scanning microscopy allows for high frame rates to capture rapid changes in mitochondrial Ca2+ in response to various drugs or reagents applied in the external solution. This protocol can be applied to measure mitochondrial Ca2+ uptake in many cell types including primary cells such as cardiac myocytes and neurons, and immortalized cell lines.

Here, we present two protocols for the measurement of mitochondrial Ca2+ influx in isolated mitochondria and cultured cells. For isolated mitochondria, we detail a plate reader-based Ca2+ import assay using the Ca2+ sensitive dye calcium green-5N. For cultured cells, we describe a confocal microscopy method using the Ca2+ dye Rhod-2/AM.

고립 된 미토 콘 드리 아 및 경작된 한 세포에 있는 미토 콘 드 리아 칼슘 유입의 분석

Ca2+ handling by mitochondria is a critical function regulating both physiological and pathophysiological processes in a broad spectrum of cells. The ...

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Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators (GECIs)

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of mitochondrial Ca2+ contributes to various pathological conditions, including neurodegeneration and apoptotic cell death in neurological diseases. Here we present a cell-type specific and mitochondria targeting molecular approach for mitochondrial Ca2+ imaging in astrocytes and neurons in vitro and in vivo. We constructed DNA plasmids encoding mitochondria-targeting genetically encoded Ca2+ indicators (GECIs) GCaMP5G or GCaMP6s (GCaMP5G/6s) with astrocyte- and neuron-specific promoters gfaABC1D and CaMKII and mitochondria-targeting sequence (mito-). For in vitro mitochondrial Ca2+ imaging, the plasmids were transfected in cultured astrocytes and neurons to express GCaMP5G/6s. For in vivo mitochondrial Ca2+ imaging, adeno-associated viral vectors (AAVs) were prepared and injected into the mouse brains to express GCaMP5G/6s in mitochondria in astrocytes and neurons. Our approach provides a useful means to image mitochondrial Ca2+ dynamics in astrocytes and neurons to study the relationship between cytosolic and mitochondrial Ca2+ signaling, as well as astrocyte-neuron interactions.

Imaging Mitochondrial Ca2+ Uptake in Astrocytes and Neurons using Genetically Encoded Ca2+ Indicators GECIs

This proctocol aims to provide a method for in vitro and in vivo mitochondrial Ca2+ imaging in astrocytes and neurons.

유전자 인코딩 된 Ca2 + 지표 (GECIs)를 사용하여 성상 세포와 뉴런에서 이미징 미토콘드리아 Ca2 + 섭취

Mitochondrial Ca2+ plays a critical role in controlling cytosolic Ca2+ buffering, energy metabolism, and cellular signal transduction. Overloading of ...

신경과학

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Determination of the Calcium Retention Capacity of Isolated Mitochondria

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Determination of the Calcium Retention Capacity of Isolated Mitochondria