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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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 ...

This method can help answer the key questions in the cytobiological field including understanding the apoptosis and mitochondria dysfunction. The main advantage of this technique is that the investigators can easily and properly conduct the real-time monitoring of the mitochondria membrane potential via rhodamine-123 within a single cell. Culture an HN4 cell line from head and neck cell squamous carcinoma as described in the text protocol.One day before transfection, plate 0.5 million cells in two milliliters per well of culture medium without antibiotics in six-well plates. Gently dilute 100 picomoles of CLIC4 small interfering RNA or scrambled small interfering RNA in 250 microliters of reduced serum media. Then dilute five microliters of liposome reagent in 250 microliters of reduced serum media and incubate for five minutes.Combine the diluted small interfering RNA with the diluted liposome reagent and mix gently. Add the mixture to each well containing cells in medium. Mix gently by rocking the plate back and forth.Incubate the cells at 37 degrees Celsius in a CO2 incubator. Replace the medium with normal growth medium four hours after transfection. Continue to incubate the cells for an additional 20 hours before proceeding with the rhodamine-123 labeling procedure.After CLIC4 small interfering RNA treatment of HN4 cells in six-well plates, use tweezers to put circular coverslips on the bottom of new 12-well plates. Pipette 150 microliters of 100 micrograms per milliliter polylysine on the middle of each coverslip inside the 12-well plates. After two minutes, thoroughly remove the polylysine by pipetter.Next, remove the culture medium of the HN4 cells inside the dish and add one milliliter of 0.25%trypsin to detach the cells. After six minutes, add two milliliters of culture medium to neutralize the trypsin. Gently mix the HN4 cells by pipetter five times and seed the cells on circular coverslips.Place the cells in an incubator at 37 degrees Celsius with 5%CO2. After eight hours of incubation, add two micromoles per liter of rhodamine-123 and gently mix the medium up and down several times to ensure even distribution in the medium. Incubate the HN4 cells for 40 minutes at 37 degrees Celsius.Use tweezers to remove the coverslips and briefly transfer them to preheated NPSS buffer to wash the remaining liquid off. Place the coverslips on the bottom of the 500 microliter stainless steel chamber with a replaceable 25 millimeter glass coverslip bottom sealed in place by an O-ring. Fix the coverslips in place by tightening the lid on the chamber.Add 450 microliters of preheated NPSS buffer to the assembled chamber. Then place the bath under the visual field of the fluorescence microscope or confocal laser scanning fluorescence microscope. Turn on the fluorescence microscope following the manufacturer's instructions.Open the imaging system software and select the new button from the command bar to begin a new experiment. Adjust the visual field and focus to find the location of the cells in the white light. Then turn off the light and push the button to close the white light.Click the focus button from the command bar and select start focusing to switch on the fluorescence. Find the location of the cells again via the microscope with a 507 nanometer excitation and 529 nanometer long-pass emission wavelength. Adjust the visual field and focus again if needed.Select a visual field containing only 20 to 30 separated HN4 cells. Once a clear visual field is achieved, click close focusing from the focus bar. Then click acquire one from the command bar to acquire one set of images.Next, click regions from the command bar and click the OK button to edit the measurement regions. To fit for different shapes of individual cells, select the trace region tool. Circle the region of one single cell and double click when finished.Repeat the procedure until all cells are circled. Click done and select save images to find the saved location. Click zero clock and quickly push F4 to start monitoring the change of fluorescence intensity.Record the real-time change of fluorescence intensity once every five seconds for five minutes. When the baseline becomes stable, add 50 microliters of NPSS mixed with 0.5 microliters of 100 millimole per liter ATP to the chamber via pipetter. Turn on the confocal laser scanning fluorescence microscope following the manufacturer's instructions and open the bundled software.Click objective under the acquire menu to select the 63X 1.4 numerical aperture oil objective lens. Observe the specimen and find a clear visual field under the light field. Push the light selection button to switch to fluorescence mode and select the correct fluorescence filters to find the location of the cell under darkness via the microscope.Push shutter to protect the specimen when the observation is finished. Under the acquire menu, adjust the suitable PMT channel and click achieve to activate the settled optical path. Click on the configuration menu and choose laser to determine the required laser type.Click live to get real-time images and make sure the visual field contains only 20 to 30 separated HN4 cells. Next, select XYT in the acquisition mode in the acquisition submenu under the acquire menu. Set five seconds for the time interval and 20 minutes for the duration.Click apply when all parameters are settled. Under the quantify menu, use the draw polyline tool to circle the recording area of each cell. Select line profile and choose start to conduct observation.Record the real-time change of fluorescence intensity for five minutes. When the baseline becomes stable, add 50 microliters of NPSS mixed with 0.5 microliters of 100 millimole per liter of ATP to the chamber via a pipetter. To perform statistical analysis using a common fluorescence microscope, open the imaging system software and select the open button from the command bar to open a saved experiment.Click regions from the command bar and click the OK button to edit the measurement regions. Select the trace region tool. Circle the region of one single cell and double click when done.Repeat the procedure until all cells have been circled. Click done and select the F4 forward button to obtain a trace showing fluorescence intensity. Once finished, click the down arrow button located on the lower right corner of the graph and click show graph data.Click OK twice and choose the log data button to open the interface of data processing software. Then click log data again to find the records of real-time fluctuation of fluorescence intensity that appear on the spreadsheet. Shown here are representative results showing ATP-induced changes of mitochondrial depolarization in the HN4 cell under different intervention as resolved by common fluorescence microscopy.The mitochondrial membrane depolarization rapidly increased following ATP treatment and gradually recovered to the normal level in both groups. Representative traces of rhodamine-123 fluorescent intensity demonstrate MMP treatment of CLIC4 small interfering RNA enhanced the mitochondrial depolarization when compared to the control group. A representative histogram further revealed that knockdown of CLIC4 enhanced the mitochondrial depolarization as compared to the control group.Here, representative confocal laser scanning microscopy results also show ATP-induced changes of mitochondrial depolarization in the HN4 cell under different intervention. The mitochondrial membrane depolarization rapidly increases following ATP treatment and gradually recovers to normal level in both groups. Representative traces of rhodamine-123 fluorescent intensity demonstrate that MMP treatment of CLIC4 small interfering RNA enhanced the mitochondrial depolarization as compared to the control group.Similarly, the representative histogram further revealed that knockdown of CLIC4 enhanced the mitochondrial depolarization as compared to the control group. After watching this video, you will have a better understanding of how to use rhodamine-123 to conduct real-time monitoring of the mitochondrial membrane potential by both normal fluorescence microscopy and confocal laser scanning microscopy. Once mastered, this technique of microscopy with rhodamine-123 can be done in less than 20 minutes for one sample if it is performed properly.While attempting this procedure, it is important to remember not to make any contact with the chamber while injection awaiting for the scanning process. Following this procedure, other methods like Western Blot and flow cytometry can be performed to answer further questions like whether apoptosis or necrosis occurred. After its development, this technique paved the way for the researchers in the field of live science to explore the mitochondrial dysfunction and find solid evidence for cell apoptosis in various living cells.Don't forget that working with rhodamine-123 can be extremely hazardous and precautions such as wearing gloves and face masks should always be taken while performing this procedure.

Research

JoVE Journal

Cancer Research

How to Study Basement Membrane Stiffness as a Biophysical Trigger in Prostate Cancer and Other Age-related Pathologies or Metabolic Diseases

Here we describe a protocol that can be used to study the biophysical microenvironment related to increased thickness and stiffness of the basement ...

Characterization of Cell Membrane Extensions and Studying Their Roles in Cancer Cell Adhesion Dynamics

The cell membrane&#39;s extension repertoire modulates various malignant behaviors of cancer cells, including their adhesive and migratory potentials. The ...

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Transfer of Manipulated Tumor-associated Neutrophils into Tumor-Bearing Mice to Study their Angiogenic Potential In Vivo

The contribution of neutrophils to the regulation of tumorigenesis is getting increased attention. These cells are heterogeneous, and depending on the ...

In Vivo Immunofluorescence Localization for Assessment of Therapeutic and Diagnostic Antibody Biodistribution in Cancer Research

Monoclonal antibodies (mAbs) are important tools in cancer detection, diagnosis, and treatment. They are used to unravel the role of proteins in ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Cell Cycle-specific Measurement of &#947;H2AX and Apoptosis After Genotoxic Stress by Flow Cytometry

The presented method or slightly modified versions have been devised to study specific treatment responses and side effects of various anti-cancer ...

Using the Chicken Chorioallantoic Membrane In Vivo Model to Study Gynecological and Urological Cancers

Mouse models are the benchmark tests for in vivo cancer studies. However, cost, time, and ethical considerations have led to calls for alternative in vivo ...