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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The authors have nothing to disclose.

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 ...

It's a messer that call answer a key question that you have in also a function of field such as ATP level and the oxygen consumption rates. The new or the one page old, this technique is that it's easy to perform and it's a lot for a special evaluation of cellular mitochondrial function. To start this experiment, seed 20, 000 HepG2 cells in a glass bottom Petri dish.Add beta-hexachlorcyclohexane or B-HCH in incubator at 37 degrees Celsius and five percent CO2 for 24 hours. Prepare one millimolar mitochondrial green fluorescence probe solution by adding anhydrous DMSO to DMEM cell culture medium. Then add one milliliter per dish of this solution to the cells in glass bottom Petri dishes.And incubate at 37 degrees Celsius for 45 minutes. After incubation, remove the mitochondrial green fluorescent probe solution from the cells. Add one milliliter per dish of freshly prepared at 37 degrees Celsius DMEM cell culture medium.To detect green fluorescence in mitochondria, observe the cells under a confocal microscope. To evaluate levels of cellular ATP, seed to 20, 000 HepG2 cells in six well plates. Add beta-HCH and incubate at 37 degrees Celsius for 24 hours.Add 200 microliters of lysis buffer to the cells in each well. Centrifuge at 12, 000 times G for five minutes at four degrees Celsius, and proceed with collecting supernatant as described in the text protocol. Add 100 microliters ATP detection buffer to the plate.Add 100 microliters of collected cell supernatant to a 96 well plate at room temperature and proceeded to detect cellular ATP by a luminometer as described in the text protocol. For assessment of mitochondrial membrane potential, grow HepG2 cells in six well plates with the addition of beta-HCH, as previously described. Collect the cells by digesting them with 0.5 milliliters 0.25%EDTA for one minute at 37 degrees Celsius.Add one milliliter DMEM to stop digestion and transfer the digested cells to 1.5 milliliter tubes. Centrifuge the tubes at 1000 times G for three minutes, and then discard the supernatant. Dilute the cell pellet with 0.5 milliliters of cell culture medium and 0.5 milliliters JC1 mitochondrial membrane potential dye.And incubate for 20 minutes at 37 degrees Celsius. Next, centrifuge the tubes at 600 times G for three minutes at four degrees Celsius, and then wash the cell pellet according to the text protocol. After centrifuging the washed pellet, remove the supernatant.And re-suspend the pellet in 0.5 milliliters of JC1 dyeing buffer. Analyze the JC1 stained cells via flow cytometry to detect green and red fluorescence. When the JC1 monomer is detected, set the excitation light to 490 nanometers and the emission light to 530 nanometers.When the JC1 polymer is detected, set the excitation light to 525 nanometers and the emission light to 590 nanometers. To analyze the oxygen consumption rate, seed HepG2 cells in a 96 well cell culture plate in a density of 4, 500 cells per 100 microliters per well. After 24 hours of incubation at 37 degrees Celsius, make sure that the cells in each well are covered with medium.Before running the OCR software, load the microplate with cartridge and the cell culture plate into an extracellular flux analyzer as described in the text protocol. Open the OCR software and in the apps drop down menu, choose cell mito stress test kit, and click on the start app button. Next, click on the run stress test button.When the cell stress test screen appears, enter the number of cells seeding per well in the cell seeding number box and the average OCR in the average at basal cell OCR box. Enter the final working concentration for each reagent and then inject the reagents. Click on the next button.When the group info screen appears, assign a group to unassigned wells by choosing a color and giving a name. Then click on the appropriate wells. Click the next button and in the stress test injection layout screen that appears, click on start to run the stress test on the analyzer.After the completed run, follow the prompts in the software and then remove and discard the cartridge and cell plate. Average intensity of mitochondrial green fluorescent staining decreased in HepG2 cells exposed to beta-HCH. This shows that there is a decrease in mitochondrial number and increase in damaged mitochondria consistent with the increase in pesticide concentration.Furthermore, ATP levels in HepG2 cells decreased with the increased concentrations of beta-HCH exposure, showing the decreased function of mitochondria in these cells. After JC1 staining for mitochondrial membrane potential, or MMP, flow cytometer showed high red green JC1 fluorescence in intact HepG2 cells. This was due to the presence of red fluorescent JC1 aggregates, which are a sign of high MMP.In cells treated with beta-HCH, red green JC1 fluorescence decreased due to a presence of green fluorescent JC1 monomers, a sign of low MMP. Finally, oxygen consumption rate also decreased with the increased concentrations of beta-HCH exposure, further showing that this pesticide impairs proper mitochondrial function. Oh, well, attempt to this procedure, it's important though, to remember to see that cells as appropriate to test it.After its development, this definitely paves a way for researches in the field of fundamental function to explore relevant organisms in the medical role associated with it. After watching this video, you should have a good understanding of how to detect an amount or quality, capacity, now member or potential under spoutage sheet function.

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7.8K 조회수.  Nanjing Medical University. ATP 수준 및 산소 소비속도와 같은 분야의 기능에 있어 도의 주요 질문에 대한 답변을 요청하는 메서입니다. 새로운 또는 한 페이지 이전, 이 기술은 수행하기 쉽고 세포 미토콘드리아 기능의 특별한 평가를 위해 많은 것입니다. 이 실험을 시작하려면, 씨앗 20, 000 HepG2 세포 유리 바닥 페트리 접시에.베타 헥사클로클로헥산 또는 B-HCH를 인큐베이터에 섭씨 37도, CO2 5%를 24시?...