Name: detection of mitochondria membrane potential to study clic4 knockdown-induced hn4 cell apoptosis in vitro
Uploaded: 2018-07-17T13:00:00
Description: Watch this Scientific Journal Video about Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro at JoVE.com

We kindly thank Mr. Chao Fang for cell culture. This work was supported by grants from the Natural Science Foundation of China (Grant No. 81570403, 81371284); Anhui Provincial Natural Science Foundation (Grant No. 1408085MH158); Outstanding Young Investigator of Anhui Medical University; Supporting Program for Excellent Young Talents in Universities of Anhui Province.

1. Cell Culture and Transfection
<ol>
	<li>Cell culture 
	Note: HN4, an HNSCC cell line, was derived from patients with HNSCC14.
	<ol>
		<li>Culture HN4 cells in Dulbecco&#39;s modified Eagle medium (DMEM, 4.5 g/L glucose) supplemented with 10% fetal bovine serum and antibiotics (100 U/mL penicillin and 100 &#181;g/mL streptomycin). Incubate cells at 37 &#176;C with 5% CO2.</li>
	</ol>
	</li>
	<li>Cell transfection
	<ol>
		<li>One day before tran...

<ol>
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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+management+of+squamous+cell+carcinoma+of+the+head+and+neck.">Advances in the management of squamous cell carcinoma of the head and neck.</a> F1000Prime Rep. 6, 44 (2014).</li><li>Behera, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+therapy+with+taxane+versus+non-taxane+containing+regimens+in+locally+advanced+squamous+cell+carcinomas+of+the+head+and+neck+(SCCHN):+a+systematic+review.">Concurrent therapy with taxane versus non-taxane containing regimens in locally advanced squamous cell carcinomas of the head and neck (SCCHN): a systematic review.</a> Oral Oncol. 50, 888-894 (2014).</li><li>Pancari, P., Mehra, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+therapy+for+squamous+cell+carcinoma+of+the+head+and+neck.">Systemic therapy for squamous cell carcinoma of the head and neck.</a> Surgical Oncology Clinics of North America. 24, 437-454 (2015).</li><li>Peretti, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+channels+in+cancer:+Focus+on+chloride+intracellular+channel+1+and+4+(CLIC1+AND+CLIC4)+proteins+in+tumor+development+and+as+novel+therapeutic+targets.">Chloride channels in cancer: Focus on chloride intracellular channel 1 and 4 (CLIC1 AND CLIC4) proteins in tumor development and as novel therapeutic targets.</a> Biochim Biophys Acta. 1848, 2523-2531 (2015).</li><li>Jentsch, T. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+structure+and+physiological+function+of+chloride+channels.">Molecular structure and physiological function of chloride channels.</a> Physiol Rev. 82, 503-568 (2002).</li><li>Ponnalagu, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Data+supporting+characterization+of+CLIC1,+CLIC4,+CLIC5+and+DmCLIC+antibodies+and+localization+of+CLICs+in+endoplasmic+reticulum+of+cardiomyocytes.">Data supporting characterization of CLIC1, CLIC4, CLIC5 and DmCLIC antibodies and localization of CLICs in endoplasmic reticulum of cardiomyocytes.</a> Data Brief. 7, 1038-1044 (2016).</li><li>Fernandez-Salas, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mtCLIC/CLIC4,+an+Organellular+Chloride+Channel+Protein,+Is+Increased+by+DNA+Damage+and+Participates+in+the+Apoptotic+Response+to+p53.">mtCLIC/CLIC4, an Organellular Chloride Channel Protein, Is Increased by DNA Damage and Participates in the Apoptotic Response to p53.</a> Mol Cell Biol. 22, 3610-3620 (2002).</li><li>Suh, K. 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It is well documented that Cl&#8722; channels are essential in maintaining the hemostasis of the internal environment and play important roles in cell proliferation and apoptosis15,16. Therefore, understanding the relationship between ion channel-targeted intervention and apoptosis is of great need and significance to find a better therapeutic approach for various cancers17. Mitochondria maintain the normal biological state of a...

Rh123 is a cationic fluorescence dye, which serves as an indicator for transmembrane potential. Rh123 is capable of penetrating the cell membrane and entering the mitochondrial matrix depending on the potential difference of inside and outside the membrane1. Apoptosis leads to damage of mitochondrial membrane integrity. The mitochondrion permeability transition pore (MPTP) will open and lead to collapse of the MMP, which in turn results in the release of Rh123 to the outside of the mitochondria. Finally, stronger green fluorescence signal will be detected under fluorescence microscope. It is well documented that depletion of the MMP and elevated membrane permeability are early signs of cell apoptosis2. Therefore, Rh123 may be applied to the detection of MMP change and the occurrence of cell apoptosis.
As the 6th most common carcinoma in the world, head and neck cancer severely deteriorates a person&#39;s health3. Although many approaches were developed in recent years, clinical outcome of treatment for patients suffering from head and neck squamous cell carcinoma (HNSCC) is still not ideal4. Exploring new therapeutic methods can improve the treatment for HNSCC5. Ion channels involving numerous biological processes display an important role in the development of different cancers6. Partial or total participation of Cl- channels are highly involved in various properties of neoplastic transformation including active migration, high rate of proliferation and invasiveness. In light of this, the CLIC, a novel protein family, has been listed as a promising class of therapeutic targets for cancer treatment6,7. Recent studies have revealed that members of the CLIC family including CLIC1, CLIC4, and CLIC5, localize to cardiac mitochondrial and the reactive oxygen species (ROS) level is upregulated by CLIC5, indicating the functional role of mitochondrial-located Cl- channels in the apoptotic response8. CLIC4, one members of the CLIC family (also known as mtCLIC, P64H1, and RS43), has been most extensively studied for its apoptotic regulation properties in cancer cells and subcellular location including Golgi, endoplasmic reticulum, and mitochondrion in human keratinocytes7,9,10. The expression profile of CLIC4 was regulated by tumor necrosis factor-&#945; (TNF-&#945;), P53, and external stimulus. Overexpression and downregulation of CLIC4 trigger an apoptotic response mainly through the mitochondrial pathway accompanied with the imbalance of Bcl-2 family members, activation of caspase cascade, and release of cytochrome C11,12,13. Therefore, MMP measurement is crucial to explore CLIC4-related apoptosis, and Rh123 serves as an ideal fluorescence indicator.
The present study describes a detailed protocol for the detection of MMP to study CLIC4 knockdown-induced apoptosis in HN4 cells. Rh123 is used as a fluorescence probe to observe the change of the MMP. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time fluctuation of the MMP may be resolved. We discuss the advantages and limitations of the application of confocal laser scanning fluorescence microscope in detail, and also compare it with other methods. This protocol also can be applied to other apoptosis-related studies.

<table border="0" cellpadding="0" cellspacing="0" style="width:1296px;" width="1294">
	<colgroup>
		<col />
		<col span="2" />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:372px;">HNSCC cells</td>
			<td style="width:183px;">ATCC</td>
			<td style="width:183px;">CRL-3241</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Polylysine</td>
			<td>Thermo Fisher Scientific</td>
			<td>P4981</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Specific siRNA for human CLIC4</td>
			<td>Biomics</td>
			<td>NM_013943</td>
			<td style="width:559px;">(accession numbers, NM_013943; corresponding to the cDNA sequence 
			5-GCTGAAGGAGGAGGACAAAGA-3) and scrambled siRNA (5 ACGCGUAACGCGGGAAUUU-3) were designed and obtained from Biomics Company</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamine 2000 Transfection Reagent&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>L3000-015</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Opti-MEM I Reduced Serum Medium, GlutaMAX</td>
			<td>Thermo Fisher Scientific</td>
			<td>51985-042</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Rhodamine 123, FluoroPure grede</td>
			<td>Thermo Fisher Scientific</td>
			<td>R22420</td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:372px;">Dulbecco&#8217;smodified Eagle medium (DMEM, 4.5 g/L glucose)</td>
			<td>Gibco</td>
			<td>11965-084</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:372px;">Fetal Bovine Serum, Qualified, Australia Origin</td>
			<td>Gibco</td>
			<td>10099141</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:372px;">Trypsin-EDTA Solution</td>
			<td>Beyotime</td>
			<td>C0201</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:372px;">Antibiotic-Antimycotic, 100X</td>
			<td>Gibco</td>
			<td>15240062</td>
			<td style="width:559px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Laser Scanning Confocal Microscopy</td>
			<td>Leica Microsystems GmbH</td>
			<td>LEICA.SP5-DMI6000-DIC</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nikon Eclipse TE300 Inverted Microscope</td>
			<td>Nikon</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Metaflour, V7.5.0.0</td>
			<td>Universal Imaging Corporation</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Leica application suite, v2.6.0.7266</td>
			<td>Leica Microsystems GmbH</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Microsoft office Excel 2007</td>
			<td>Microsoft</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sigma Plot 12.5</td>
			<td>Systat Software</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Attofluor Cell Chamber</td>
			<td>Thermo Fisher Scientific</td>
			<td>A7816</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of mitochondria membrane potential to study clic4 knockdown-induced hn4 cell apoptosis in vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of nuclear apoptotic characteristics, including chromatin condensation and DNA breakage. Once the MMP collapses, cell apoptosis will initiate irreversibly. A series of lipophilic cationic dyes can pass through the cell membrane and aggregate inside the matrix of mitochondrion, and serve as fluorescence marker to evaluate MMP change. As one of the six members of the Cl- intracellular channel (CLIC) family, CLIC4 participates in the cell apoptotic process mainly through the mitochondrial pathway. Here we describe a detailed protocol to measure MMP via monitoring the fluorescence fluctuation of Rhodamine 123 (Rh123), through which we study apoptosis induced by CLIC4 knockdown. We discuss the advantages and limitations of the application of confocal laser scanning and normal fluorescence microscope in detail, and also compare it with other methods.

In the present study, Rh123 was applied to detect the MMP. Initially, HN4 cells were cultured for the following fluorescence staining experiments. Tweezers were used to put circular coverslips on the bottom of 6-well plates (Figure 1A). The coverslips were coated with polylysine for 5 min and then the polylysine removed via pipettor (Figure 1B). Then HN4 cells were trypsinized and seeded on the c...

Watch this Scientific Journal Video about Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro at JoVE.com

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Here we present a detailed protocol for the application of rhodamine 123 to identify the mitochondrial membrane potential (MMP) and study CLIC4 knockdown-induced HN4 cell apoptosis in vitro. Under common fluorescence microscope and confocal laser scanning fluorescence microscope, the real-time change of the MMP was recorded.

Detection of Mitochondria Membrane Potential to Study CLIC4 Knockdown-induced HN4 Cell Apoptosis In Vitro

Depletion of the mitochondrial membrane potential (MMP, &#916;&#936;m) is considered the earliest event in the apoptotic cascade. It even occurs ahead of ...

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