Strategies for Tracking Anastasis, A Cell Survival Phenomenon that Reverses Apoptosis

The overall goal of this procedure is to detect and track reversal of the apoptotic cell death process and cultured cells by live cell microscopy. Healthy cells spread on the substrate and contain tubular filamentous mitochondria around the nucleus. However, when cells commit suicide through apoptosis, the dying cells display unique morphological features to detect and track reversal of apoptosis.

Cells are plated into glass bottom cell culture dishes. The next step is to perform live cell staining to label mitochondria and nuclei. This allows for detection of morphological features of healthy and apoptotic cells.

By live cell microscopy, the labeled healthy cells are then exposed to a death stimulus to induce apoptosis. When apoptosis is detected, the dying cells are washed and then incubated with fresh cell culture medium to allow cell recovery, live cell imaging is used to detect and track reversal of apoptosis and its consequences such as cell repairing and survival as shown by cell morphological recovery and migration respectively, Respectively. Apoptosis is a type of programmed cell death that pay essential role to eliminate unwant or dangerous cells in our body.

These cell synthesized process is generally assumed to be irreversible because once it has started cell dies certainly afterward, however, we discovered that in fact, dying cell can reverse initiate the cell death process and then survive and derive even. They have possible critical steps that has been forced to be the point of low return, such as cyto C, release, case based activation, nuclear fragmentation, and apoptotic body formation. After consulting Greek experts, we adopt a term, an stasis, which means rising to life.

To describe the phenomenon of reversing apoptosis and other cell death processes, anthesis is difficult to detect because the cell that reverse the apoptosis look like normal heavy cells. However, by continuous live cells microscopy, we can detect anastasis to occur in large portion of cells. Mastering this technique is important because it allow us to study the recreation of anthesis.

We proposed that that anthesis might heal damage the tissue, but it could also allow damage cells to survive and form cancers. Therefore, identifying mechanisms that control anesthesias might provide us new approaches to treat in injectable diseases such as cancer, heart failure, and degeneration by mediating cell death and survival To detect and track anastasis. After apoptotic events, time-lapse live cell microscopy is performed to track which cells can reverse apoptosis after transient cell death induction.

To begin, add one milliliter of poly de lycine solution to a 35 millimeter cell culture dish with a thinner glass bottom. Following a one minute incubation to coat the glass surface, aspirate the solution and wash the glass with two milliliters of phosphate buffered saline To prepare cells first trypsin eyes, heela cells at 80 to 90%confluence After the cells have detached, add 10 milliliters of 37 degrees Celsius medium to suspend the cells. Then transfer the cell suspension solution to a 10 milliliter sterile tube to centrifuge at 160 times G for one to two minutes.

Following the spin, carefully aspirate the medium. Do not disturb the cell pellet at the bottom of the tube. Proceed to resuspend the cells with 10 milliliters of cell culture medium by gentle pipetting.

Then see two milliliters of the suspended cells on 35 millimeter pre-coated glass. Bottom cell culture dishes. Incubate the cells in a humidified incubator at 37 degrees Celsius with 5%carbon dioxide for a day.

Adjusting cell numbers may be necessary so that cells can achieve 80%co fluency at the time of imaging. Mitochondrial and nuclear fragmentation are the morphological hallmarks of apoptosis. To visualize mitochondria and nuclei perform staining shortly before live cell imaging.

Begin the staining protocol by first adding two microliters of mitochondrial stain. MIT tracker red 50 micromolar stock solution, and two microliters of DNA stain hooked 3 3 3 4 2 10 milligrams per milliliter stock solution in a sterile micro centrifuge tube, add one milliliter of the conditioned culture medium from the cell culture dish to the micro centrifuge tube and mix well with the stains by pipetting. Then add the mixture back to the culture dish.

After incubating the dish at 37 degrees Celsius with 5%carbon dioxide for 20 minutes, aspirate the medium and wash the stained cells three times with one milliliter of warm cell culture, medium or phosphate buffered saline. Then incubate the cells with cell culture medium at 37 degrees Celsius with 5%carbon dioxide for an additional five minutes. Avoid prolonged staining time as non-specific background signal of the stain cells could increase due to over staining.

Following incubation, aspirate the medium, wash the cells again, and then continue to incubate the cells with two milliliters of cell culture medium at 37 degrees Celsius with 5%carbon dioxide. The microscope components must reach thermo equilibrium to avoid the drift of focus and shift of the XY plane due to the thermal expansion and contraction of the components. To ensure thermo equilibrium is achieved, turn on the environmental control chamber and stage incubator to prewarm up the microscope to 37 degrees Celsius at least two hours before imaging starts.

Then turn on the carbon dioxide regulator to supply 5%carbon dioxide to the environmental control chamber. When the carbon dioxide supply is not available, carbon dioxide independent medium can be used for supporting cell growth. Prior to imaging, place a drop of immersion oil directly on a 40 x objective with a 1.4 numerical aperture.

Then gently position the culture dish with stained cells onto the 37 degrees Celsius stage incubator. Cover the dish with transparent cold foil to prevent loss of water by evaporation and contamination of the sample to perform differential interference contrast microscopy. Do not use the plastic culture dish cover as plastic can disturb polarity of light.

Next, locate and focus on a group of healthy cells that spread on the glass surface with tubular mitochondria and round nuclei. Choose the cells at or near the center of the culture dish to ensure that the cells are at the same focal plane once in place, take a quick exposure by confocal microscopy to determine the minimal laser intensity and exposure time to obtain clear images of the cells and their organelles. This step is critical.

As laser light is phototoxic to the cells, optimization and adjustment are necessary To eliminate focus drift, use a continuous automatic focus drift compensation device that uses an infrared light emitting diode. Alternatively, the focus plane can be corrected manually. When the cells and focal plane are identified, optimize all parameters and take a test image before starting the live cell imaging.

Then start live imaging to image the healthy cells with one minute intervals for 10 cycles to induce apoptosis. Remove the cell medium with a transparent pipette. Apply a cell death stimulus that is premixed with a 37 degrees Celsius cell culture medium to the dish.

Continue to cover the dish with transparent cult foil. During time-lapse imaging following application of the cell death stimulus, adjust the time interval to image the treated cells to detect apoptosis by capturing morphological hallmarks of apoptosis such as plasma membrane blobbing, mitochondrial fragmentation, nuclear condensation and fragmentation, cytoplasmic condensation, cell shrinkage, and apoptotic body formation. When apoptosis is observed, remove the medium with the cell death stimulus and apply two milliliters of fresh cell culture medium to incubate the cells.

Continue the time-lapse imaging to observe morphological recovery of the cells and the consequence of anastasis. After removal of a cell death stimulus dying human lung cancer, H 4 46 cells can reverse apoptosis. Although the cells display hallmarks of apoptosis such as cell shrinkage, membrane blabbing, mitochondrial fragmentation, nuclear condensation, and fragmentation and apoptotic, body formation after anastasis cell division can be abnormal.

In this example, instead of two dotter nuclei, three major nuclei are formed after the cell division with formation of micro nuclei indicating genetic alterations in the dotter cells. This suggests that anastasis could be mutagenic after exposure of a cell to a cell death stimulus. Pro apoptotic factors translocate to mitochondria to trigger the release of cytochrome C, which leads to assembly of a protosome and cast based activation for cell demolition and then cell death shown.

Here are HELOC cells expressing GFP tagged cytochrome C, which localizes to mitochondria after induction of apoptosis. Cytochrome C is released into the cytosol. However, after the cells are washed and then incubated with fresh medium cytosol, ex cytochrome C reduces back to the normal levels indicating that anastasis can occur after cytochrome C release.

Shown here are hela cells expressing the CA based biosensor N-E-S-D-E-V-D-Y-F-P-N-L-S, which first localizes to the cytosol after apoptosis Induction as expected. Y-F-P-N-L-S translocates to the nucleus due to the cleavage of DEVD by activated cast bases. However, after the death stimulus is washed off and the cells are incubated with fresh medium, Y-F-P-N-L-S remains in the nucleus while cells regain normal morphology indicating that reversal VA ptosis can occur after cast base activation.

After watching this video, you should have good understanding of how to use live cells imaging to detect anthesis and its consequence. In this demonstration, we use ethanol as ATO inducer. At the same time, cell can reverse cell death process that is induced by other death stimuli such as teso DMSO and sporin Fluorescent light is photo toic to the cells so that it is critical to minimize the nights that heat a sample during the live cells imaging.

The process of apoptosis and nightly anastasis depends on temperature sensitive and sematic activities. Therefore, it is important to keep the cells at the same temperature for of the experiment to ensure repeatable results.