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.
Depletion of the mitochondrial membrane potential (MMP, ΔΨm) 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.
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'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-α (TNF-α), 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.
1. Cell Culture and Transfection
2. Rh123 Labeling
Note: Rh123 was utilized to measure the MMP in HN4 cells1.
3. Detection of the MMP
4. Statistical Analysis
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 coverslips placed in the bottom of 6-well plates. Next, the appropriate amount of Rh123 was added and mixed well (Figure 1C-D). After washing with the preheated NPSS, the coverslip was assembled into the viewing chamber. The appropriate amount of NPSS was added for the following experiment (Figure 1E).
After the procedure of Rh123 staining, both the common fluorescence microscope and confocal microscope were used to record the real-time Rh123 fluorescence with different steps and software. For the common fluorescence microscope, the imaging software was turned on to create a new experiment. The shutter was opened to show the green fluorescence light and the focus and location were adjusted to obtain an appropriate visual field. After taking a cell image, the shape trace tools were used to draw the region of every single cell and then record the fluorescence change. Once the baseline became stable, ATP was added into the chamber to trigger mitochondrial membrane depolarization. In the experiment of confocal laser scanning fluorescence microscope, the bundled software was opened and a suitable optical path was set. After the argon light source was chosen, we adjusted the focus and position of the chamber to achieve a clear visual of the cell shapes on screen. The "xyt" scan mode was selected and the "Draw polygon" tool was applied to circle the cell region to record the fluorescence change within every single cell. To analyze the raw data, F0 was the value of fluorescence intensity before ATP application and F1 was the maximum value of fluorescence intensity after ATP application. F1/F0 was used to assess the mitochondrial membrane depolarization (Figure 2 and Figure 3). From Figure 4B and Figure 5B, the levels of fluorescent intensity were elevated following the treatment of ATP and decreased back to the baseline after about 10 min. Figure 4C and Figure 5C clearly demonstrate that the peak of fluorescence intensity of Rh123 was higher in the group under the CLIC4 siRNA treatment than that in control group. Figure 4A and Figure 5A show representative images of the fluorescent change in each stage. Compared with the common fluorescence microscope, the confocal microscope obtained a higher quality of image, showing cell nucleus and mitochondria. However, when comparing the data from the two methods, no significant differences were found. Results from fluorescence and confocal microscope demonstrated that knockdown of CLIC4 enhanced ATP-induced mitochondrial membrane depolarization in HN4 cells (Figure 4 and Figure 5).
Figure 1. Rh123 labeling. (A) Tweezers were used to put circular coverslips on the bottom of 6-well plates. (B) Coating coverslips with polylysine via pipettor for 5 min and finally suction back into pipettor to remove the polylysine. (C) HN4 cells were seeded on the coverslips on the bottom of 6-well plates. (D) Adding a suitable amount of Rh123 and mixing well. (E) Washing the coverslips with preheated NPPS and assembling the chamber with a suitable amount of NPSS for the following experiment. Please click here to view a larger version of this figure.
Figure 2. Steps to monitor real-time fluctuation of Rh123 fluorescence by common fluorescence microscope. (A) Open a new experiment. (B) Start focusing and choose a suitable visual field under fluorescence. (C) Using the "Regions" tool from the Command Bar to edit observation regions. (D) Click "Zero clock" and quickly push F4 to start monitoring the change of fluorescence intensity. (E) Adding ATP (100 µmol/L) to trigger mitochondrial membrane depolarization after the baseline becomes stable. (F) Once the experiment is finished, click the down arrow located on the lower right corner of the graph to export data. Please click here to view a larger version of this figure.
Figure 3. Steps to monitor real-time fluctuation of Rh123 fluorescence by confocal laser scanning fluorescence microscope. (A) Select the PMT channel and set a suitable wavelength range under the "Acquire" menu. (B) Select the correct laser type from "Current available laser" and set its power. (C) Choose the "xyt" observing mode and set a series of recording parameters. (D) Choose the tools "Stack Profile" and "All in One" from "Sort charts by Channels and ROIs." (E) Use the tool "Draw polygon" to circle the cell region for the real-time data. (F) Export the values of fluorescent intensity to data processing software. Please click here to view a larger version of this figure.
Figure 4. Effect of CLIC4 knockdown on ATP-induced mitochondrial membrane depolarization resolved by common fluorescence microscope in HN4 cells. (A) Representative images of fluorescence fluctuation for Rh123. Representative traces (B) and summarized data (C) showing ATP (100 µmol/L)-induced changes in the HN4 cell MMP. The change in the membrane potential was indicated by the ratio of the fluorescence intensity before and after the application of ATP. The ratio increase represents the membrane potential depolarization. The HN4 cells were transfected with CLIC4siRNA (CLIC4 siRNA) or scrambled siRNA (Con siRNA). Values are shown as the mean ± SEM. n = 5. *P <0.05 vs. control (Con siRNA) group. Please click here to view a larger version of this figure.
Figure 5. Effect of CLIC4 knockdown on ATP-induced mitochondrial membrane depolarization resolved by confocal laser scanning fluorescence microscope in HN4 cells. (A) Representative images of fluorescence fluctuation for Rh123. Representative traces (B) and summarized data (C) showing ATP (100 µmol/L)-induced changes in the HN4 cell MMP. The change in the membrane potential was indicated by the ratio of the fluorescence density before and after the application of ATP. The ratio increase represents the membrane potential depolarization. The HN4 cells were transfected with CLIC4 siRNA (CLIC4 siRNA) or scrambled siRNA (Con siRNA). Values are shown as the mean ± SEM. n = 5. *P <0.05 vs. control (Con siRNA) group. Please click here to view a larger version of this figure.
It is well documented that Cl− 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 cell and its function highly relates to its membrane permeability and transmembrane potential. Apart from cancer, accumulating neuropathological investigations have documented that mitochondrial abnormalities contribute to myopathy, cardiovascular disease, and neurological symptoms including sensorineural deafness, cerebellar ataxia, dementia, and epilepsy18,19. All these have spurred the research in mitochondrial-targeted intervention with which to ameliorate clinical treatment. Rh123, a mitochondrion-targeted fluorescence dye, can penetrate the cell membrane, and serves as a universal fluorescence probe to detect MMP. In the early state of cell apoptosis, opening of MPTP elevates the mitochondrial membrane permeability allowing Rh123 to be released outside the mitochondria and finally stronger green fluorescence signal may be detected. Under this situation, bilateral ions distribute freely and lead to the rapid drop of MMP causing decoupling of the electron transport chain and decreased ATP production, which severely affects the normal energy supply of cells. Additionally, opening of MPTP triggers the outflow of pro-apoptotic bioactive compounds including Cyt C, apoptosis inducing factor, apoptotic protease activating factor-1, and ROS, and finally leads to irreversible apoptosis20,21.
ATP may induce mitochondrial membrane depolarization as demonstrated by elevated fluorescence of Rh123. By comparing ATP-induced mitochondrial membrane depolarization of cells under different treatments, it is possible to decipher the biological function of the change in mitochondria and the degree and state of apoptosis22. Both common fluorescence microscope and confocal laser scanning fluorescence microscope can achieve real-time monitoring of MMP via recording the fluctuation of fluorescence intensity of Rh123, but advantages and shortcomings of both methods need to be considered. Compared with the common fluorescence microscope, the images captured by confocal laser scanning fluorescence microscope have higher resolution and quality. Therefore, more subcellular details can be visualized. Additionally, it diminishes the impacts of the light cross-talk when multiple fluorescence dyes are used. Hence, the confocal microscope can precisely record the real-time fluorescence fluctuation of Rh123 and reflect the real cellular bioactivity. However, our methods to monitor MMP need continuously recording for a relative long time and a series of cytotoxins, including single oxygen and ROS, may be produced under the laser radiation leading to cell damage23. Moreover, high laser intensity usually causes fading of fluorescence dyes during the continuous scanning24. By contrast, the common fluorescence microscope cannot take images with high quality like the confocal microscope, but it does not have the aforementioned adverse effects and thus makes long time recording possible. By comparing our data from both common fluorescence microscope and confocal microscope, no obvious difference could be found, which indicates that the common fluorescence microscope is competent enough for monitoring the fluctuation of MMP, as well as cost-effective and more convenient data analysis. HN4 cells were seeded on coverslips before the experiment. Although polylysine is applied to enhance the cellular attachment, a small fraction of cells still detached from the coverslips and rough operation leads to the impairment of cell viability. Besides, adding Rh123 or ATP into the chamber, care should be taken to not touch the chamber and coverslips.
Besides Rh123, DioC6, JC-1, and tetramethyl rhodamine methyl ester (TMRM) are fluorescence dyes that may be used to detect the MMP. Instead of real-time recording via microscope, flow cytometry also can accomplish this task. However, recording the real-time change of fluorescence intensity with the microscope is more suitable to discover the cellular bioactivity and generate intuitionistic images, giving more reliable results.
The authors have nothing to disclose.
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.
HNSCC cells | ATCC | CRL-3241 | |
Polylysine | Thermo Fisher Scientific | P4981 | |
Specific siRNA for human CLIC4 | Biomics | NM_013943 | (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 |
Lipofectamine 2000 Transfection Reagent | Thermo Fisher Scientific | L3000-015 | |
Opti-MEM I Reduced Serum Medium, GlutaMAX | Thermo Fisher Scientific | 51985-042 | |
Rhodamine 123, FluoroPure grede | Thermo Fisher Scientific | R22420 | |
Dulbecco’smodified Eagle medium (DMEM, 4.5 g/L glucose) | Gibco | 11965-084 | |
Fetal Bovine Serum, Qualified, Australia Origin | Gibco | 10099141 | |
Trypsin-EDTA Solution | Beyotime | C0201 | |
Antibiotic-Antimycotic, 100X | Gibco | 15240062 | |
Laser Scanning Confocal Microscopy | Leica Microsystems GmbH | LEICA.SP5-DMI6000-DIC | |
Nikon Eclipse TE300 Inverted Microscope | Nikon | N/A | |
Metaflour, V7.5.0.0 | Universal Imaging Corporation | N/A | |
Leica application suite, v2.6.0.7266 | Leica Microsystems GmbH | N/A | |
Microsoft office Excel 2007 | Microsoft | N/A | |
Sigma Plot 12.5 | Systat Software | N/A | |
Attofluor Cell Chamber | Thermo Fisher Scientific | A7816 |