Name: experimental protocol for detecting mitochondrial function in hepatocytes exposed to organochlorine pesticides
Uploaded: 2020-09-16T12:30:00
Description: Watch this Scientific Journal Video about Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides at JoVE.com

This work was supported by the National Natural Science Foundation of China (Grant Nos. 81573174, 81570574); the Outstanding Youth Fund of Jiangsu Province (SBK2014010296); the Research Project of Chinese Ministry of Education (213015A); the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), the Flagship Major Development of Jiangsu Higher Education Institutions; and the Open Project Program of the State Key Laboratory of Environmental Chemistry and Ecotoxicology (KF2015-01).

All experiments and the experiment protocols were performed in accordance with relevant guidelines and regulations and approved by the local Ethical Committee of Nanjing Medical University.
1. Mitochondrial ultrastructure by TEM
<ol>
	<li>Collecting HepG2 cells
	<ol>
		<li>Seed HepG2 cells in 100 mm dishes. Store at 37 &#176;C and 5% CO2.</li>
		<li>Digest cells with 0.25% EDTA in 1.5 mLEP tube.</li>
		<li>Centrifuge at 1000 x g for 3 min at room temperature (RT). Discard...

<ol>
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Critical to the success of the detection protocol is the use of a variety of experimental methods that have been covered the study from phenotype to mechanism. In this study, HepG2 cells were cultured in DMEM with penicillin and streptomycin and 10% fetal bovine serum. When cells reached 40-50% con&#64258;uence, &#946;-HCH (0, 10, 100 ng/mL) were added and incubated for 24 h. We firstly used TEM which showed the ultrastructural changes in hepatocyte caused by the representative OCPs, &#946;-HCH, showing the impairment of...

The effects of organochlorine pesticides (OCPs) on health, e.g. reproductive interference, immunological toxicity, metabolic changes have been previously studied1,2,3. The methods to detect cellular metabolism and find out mitochondrial dysfunction have enabled scientists to understand the role of mitochondrial function (i.e., mitochondrial STAT3 levels, lactate, pyruvate, lactate-to-pyruvate ratio, coenzyme Q10, mitochondrial proton leak, bioenergetics, biogenesis, and dynamics) in areas such as aging, obesity, diabetes, cardiovascular function, cancer, and safety toxicity4,5,6,7. In this paper, we describe the methods on assessing mitochondrial dysfunction caused by OCPs.
We exposed HepG2 cells to &#946;-hexachlorocyclohexane (&#946;-HCH), one representative OCPs, for 24 h at a dose equivalent to internal exposure of human. Firstly, TEM was applied to observe the ultrastructure of hepatocytes, such as nuclei, mitochondria and endoplasmic reticulum8. Compared with ordinary microscopes, TEM enables to explore the 2D and 3D ultra-structure of cells and cell components (cell lines or tissue), the morphology, chemical composition, as well as function of natural or artificial materials that play a pivotal role in modern science and technology. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5&#39;-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP)in HepG2 cells incubated with &#946;-HCH. Mito-tracker green is a mitochondria green fluorescent probe that can be used for live cell mitochondrial-specific fluorescent staining. The mitochondria in hepatocytes were stained by mito-tracker green solution and mitochondrial fluorescence intensity, number and pattern were observed with a confocal microscopy9. Mitochondrial green fluorescent probe can be used to stain live cells. Compared with rhodamine 123 or JC-1, mitochondrial green fluorescent probe does not depend on mitochondrial membrane potential for mitochondrial staining. ATP levels were determined by a luciferase-luciferin kit and normalized by protein concentration. ATP assay kit can be used to detect ATP levels in common solutions, cells or tissues. This kit is based on firefly luciferase catalyzed by fluorescein to generate fluorescence, when ATP is required to provide energy. When the firefly luciferase and fluorescein are excessive, in a certain concentration range, the generation of fluorescence is proportional to the concentration of ATP. In addition, this kit has been specially designed to optimize the chemiluminescence of ATP. ATP, as the most important energy molecules, plays an important role in the various physiological or pathological processes of cells. Changes in ATP levels can reflect defects in cell function, especially mitochondrial energy production. Usually under apoptosis, necrosis or in some toxic state, cellular ATP levels reduced 10. MMPs assay kit with JC-1 is a kit that uses JC-1 as a fluorescent probe to detect cells, tissues or purified MMPs quickly and sensitively. It can be used for early detection of apoptosis. The cationic dye JC-1 is a fluorescent probe used to detect the MMP which can be analyzed under flow cytometry indicated by changes of green and red fluorescence ratio. When the MMP is high, JC-1 is aggregated in the matrix of the mitochondria to form a polymer (J-aggregates), which produces red fluorescence. When the MMP is low, JC-1 cannot accumulate, and forms monomer which produces green fluorescence11. So the ratio of red and green fluorescence can reflect the level of MMP. The OCR of cells is a crucial indicator of normal cellular function12. It is regarded as a parameter to research mitochondrial function. Cell mito stress test kit provides a stable method for analyzing key parameters of mitochondrial function. The kit provides quality control and predictive reagents as well as a standard method for performing cell mitochondrial stress test. It can be used to detect all cell types, including primary cells, cell lines, suspended cells, and also for islets, nematodes, yeasts and isolated mitochondria.
OCR measurement can provide valuable insight into the physiological status or alterations of cells. It was determined with an extracellular flux analyzer to detect breathing baseline, proton leak, maximal respiratory, ATP turnover and reserve capacity. In brief, after baseline measurements of OCR, OCR was detected after sequentially adding to oligomycin (ATP Coupler), FCCP (mitochondrial oxidative phosphorylation uncoupler) and antimycin A/rotenone (an inhibitor of oxygen consumption)per well.
In an effort to facilitate the development of more specific protocol for detecting mitochondrial function in hepatocytes in vitro, we present here experiments by TEM, confocal microscopy, luminometer, flow cytometry and extracellular flux analyzer with future application in studying mitochondria damage related adverse outcomes.

<table border="0" cellpadding="0" cellspacing="0" style="width:756px;" width="755">
	<tbody>
		<tr height="21">
			<td>Transmission electron microscope&#160;</td>
			<td>FEI</td>
			<td>Tecnai G2 Spirit Bio TWIN</td>
			<td>High-contrast, high-resolution imaging, Low-dose observation and imaging, Low-temperature observation, Outstanding analytical performance, Automation for convenience and performance</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:205px;">Mito-Tracker Green</td>
			<td style="width:69px;">Beyotime</td>
			<td style="width:161px;">C1048</td>
			<td style="width:320px;">Mito-Tracker Green is a mitochondrial green fluorescent probe that can be used for live cell mitochondrial-specific fluorescent staining.</td>
		</tr>
		<tr height="21">
			<td height="105" style="height:105px;width:205px;">Laser scanning confocal microscope</td>
			<td style="width:69px;">Zeiss</td>
			<td style="width:161px;">700B</td>
			<td style="width:320px;">The design is compact, stable, light path is the shortest, high light precision, creative technology and sophisticated scanning technology together to&#160; produce a perfect 3-dimensional specimen image.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:205px;">Enhanced ATP Assay Kit</td>
			<td style="width:69px;">Beyotime</td>
			<td style="width:161px;">S0027</td>
			<td style="width:320px;">Enhanced ATP Assay Kit can be used to detect ATP (adenosine 5&#39;-triphosphate) levels in common solutions, cells or tissues. Cells and tissue samples can be split to complete the sample preparation, detection sensitivity up to 0.1nmol / L, chemiluminescence can be sustained for 30 minutes.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:205px;">Luminometer</td>
			<td style="width:69px;">Berthold</td>
			<td style="width:161px;">Centro LB 960</td>
			<td style="width:320px;">Luminometer is chemiluminescence detector, the test sample itself can be light, do not need to stimulate. Luminometer is the instrument that detects chemiluminescence.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:205px;">BCA Protein Assay Kit</td>
			<td style="width:69px;">Beyotime</td>
			<td style="width:161px;">P0012</td>
			<td style="width:320px;">BCA Protein Assay Kit is one of the most commonly used methods for detecting protein concentrations.</td>
		</tr>
		<tr height="21">
			<td height="42" style="height:42px;width:205px;">Multimode reader</td>
			<td style="width:69px;">TECAN</td>
			<td style="width:161px;">InfiniteM200</td>
			<td style="width:320px;">Multimode reader be used to detect protein consentration.</td>
		</tr>
	</tbody>
</table>

experimental protocol for detecting mitochondrial function in hepatocytes exposed to organochlorine pesticides

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by environmental organochlorine pesticides (OCPs) in hepatocytes. HepG2 cells were exposed to &#946;-hexachlorocyclohexane (&#946;-HCH) for 24 h at an equivalent dose of internal exposure in general population. Ultrastructure in hepatocytes was examined by transmission electron microscopy (TEM) to show the damage of mitochondria. Mitochondrial function was further evaluated by mitochondrial fluorescence intensity, adenosine 5'-triphosphate (ATP) levels, oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP) in HepG2 cells incubated with &#946;-HCH. The mitochondria fluorescence intensity after stained by mitochondrial green fluorescent probe was observed with a fluorescence microscopy. The luciferin-luciferase reaction was used to determine ATP levels. The MMP was detected by the cationic dye JC-1 and analyzed under flow cytometry. OCR was measured with an extracellular flux analyzer. In summary, these protocols were used in detecting mitochondrial function in hepatocytes with to investigate mitochondria damages.

The mitochondria cristae of HepG2 cells exposed to &#946;-HCH were markedly damaged. Scattered mitochondria were mildly to markedly expanded, irregularly shaped, and mitochondrial ridge disappeared with relatively abnormal mitochondrial architecture (Figure 1).
Average mitochondrial green fluorescence intensity, which represents the mitochondria, decreased in HepG2 cells exposed to &#946;-HCH (<stro...

Watch this Scientific Journal Video about Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides at JoVE.com

Experimental Protocol for Detecting Mitochondrial Function in Hepatocytes Exposed to Organochlorine Pesticides

Understanding the influence of environmental organochlorine pesticides (OCPs) on mitochondrial function in hepatocytes is important in exploring the mechanism of OCPs causing metabolic disorders. This paper presents detailed methods on detecting hepatic mitochondrial function.

This paper presents detailed methods on detecting hepatic mitochondrial function for a better understanding the cause of metabolic disorders caused by ...

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