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

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.