Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells

Flow cytometry is a powerful technique that allows multiparametric analysis of single cells. It can identify biomarkers of early and late apoptosis accurately and rapidly by analyzing millions of cells. Multiple cell molecules are stained and simultaneously detected.

In particular, bench-top flow cytometry permits analysis and interpretation by non-expert flow cytometrists. Demonstrating the procedure will be Esther Zhou, a postgraduate student from the vitamin D cancer group. Begin by growing the cell cultures in a humidified CO2 environment at 37 degrees Celsius with 5%CO2.

Ensure that cell culture is approximately 70%confluent in the parent flask before passaging cells for experiments. Remove the medium from the flask and wash the cells with 1X PBS. Then add one milliliter of trypsin to detach the cells.

After the cells begin to detach, neutralize the trypsin by adding approximately five milliliters of fresh culture medium supplemented with 10%fetal calf serum before counting the cells. Seed cells at 15, 000 cells per milliliter in three milliliters of cell culture medium in a six-well cell culture plate. Incubate the culture plates overnight at 37 degrees Celsius with 5%CO2 to allow the attachment of the cells to the bottom of the well.

The next day, treat the experimental cultures with 100 nanograms per milliliter of actinomycin D and the medium and solvent controls with fresh culture medium and DMSO, respectively, for 24 hours. Remove the medium from the wells and collect the spent medium in a 15-milliliter tube. Wash the cells with 1X PBS.

Add 500 microliters of trypsin to detach the cells. Gently back-pipette one milliliter of medium two to three times to mechanically dislodge remaining cells still attached to the bottom of the well. Add the solution from the wells into a 15-milliliter tube and then add five milliliters of fresh culture medium to neutralize the trypsin.

Pipette 450 microliters of reagent containing DNA binding dyes in a microcentrifuge tube. Add 50 microliters of the cell suspension to the microcentrifuge tube. Incubate the tube at room temperature for five minutes.

Enumerate the cells by flow cytometry. Dilute all samples to a concentration of one times 10 to the sixth cells per milliliter with cell culture medium before proceeding to the apoptosis assays. To detect the externalization of phosphatidylserine using an annexin V, add 100 microliters of cell suspension into a microcentrifuge tube.

To that, add 100 microliters of a 1:1 mixture of fluorescent conjugated annexin V and 7-AAD reagent. Incubate at room temperature for 20 minutes, protected from light. For analysis of mitochondrial membrane depolarization, start by centrifuging 100 microliters of the cell suspension at 300 times G for five minutes and then discard the supernatant.

Resuspend the cells in one milliliter of 1X PBS. Add 100 microliters of TMRE staining solution to each sample and mix the suspension by gentle back-pipetting. Wrap the samples in clean aluminum foil to protect them from light and incubate the cells for 20 minutes in a humidified CO2 environment at 37 degrees Celsius.

At the end of the incubation, add 7-AAD working solution to the sample and mix it. To detect activated terminal caspase-3 and 7, begin by diluting the DEVE-bound DNA binding peptide in DMSO to a ratio of 1:8 with sterile 1X PBS to make the caspase detection working solution. Store the solution on ice or add two to eight degrees Celsius, protected from light.

Add two microliters of a 7-AAD stock solution to 148 microliters of 1X PBS to make the 7-AAD working solution. Store the solution on ice or at two to eight degrees Celsius, protected from light. Centrifuge 50 microliters of the cell suspension for five minutes at 300 times G and discard the supernatant.

Resuspend the cells in 50 microliters of 1X PBS, followed by five microliters of the caspase detection working solution and mix thoroughly. Loosen the cap of the tubes and incubate for 30 minutes in a humidified CO2 environment at 37 degrees Celsius, protected from light. Add 150 microliters of the 7-AAD working solution to each sample and mix.

Incubate for five minutes at room temperature, protected from light. For detection of double-strand DNA breaks and total DNA damage, first centrifuge 50 microliters of the cell suspension for five minutes at 300 times G and discard the supernatant. Resuspend the cells in 500 microliters of 1X PBS and add 500 microliters of a formaldehyde-based fixation buffer and mix.

Incubate the samples on ice for 10 minutes. Centrifuge for five minutes at 300 times G and discard the supernatant. Resuspend the cells in 90 microliters of 1X PBS in a microcentrifuge tube.

Add 10 microliters of the antibody solution to the microcentrifuge tube. Incubate at room temperature for 30 minutes in the dark. Add 100 microliters of 1X PBS and centrifuge for five minutes at 300 times G.Discard the supernatant and resuspend the cells in 200 microliters of 1X PBS.

Mix the sample by gently back-pipetting before loading the sample onto the flow cytometer. Verify the analytical performance of the flow cytometer by running the instrument's system check kit and do not proceed with running samples until all checks are completed and passed. First, load a negative control sample onto the flow cytometer and select Run, Adjust Settings, so that the instrument begins aspirating the sample and provides a real-time preview of detected events.

Using the live preview, adjust the thresholds for fluorescence and cell size. Draw a rectangular gate around the cell population, drag the threshold marker to exclude cellular debris, and select Next, Set Apoptosis Profile to proceed. Tap and drag the quadrant markers to separate the cell populations and to plot detected events in real-time.

Use these plots to guide the end-user on the appropriate placement of the quadrant markers. Select Next, Verify Settings to proceed so that the instrument displays a summary of the settings. After reviewing the settings, select Next, Finish Settings to apply these settings for all samples within the experiment.

Mix the first sample by gently back-pipetting and load the first sample onto the flow cytometer. Select Next, then name the sample and select Run so that the system begins running the sample. If required, perform fine-tuning of gates or quadrant markers post-acquisition.

Locate the experiment that needs adjustment by navigating the system's file browser and open the experiment. Tap on the thumbnail preview of the plot to enlarge it. To adjust the quadrant markers, click on the intersection of the vertical and horizontal lines to move the markers as they are, and adjust the angle of either line by clicking on the line and drag the handle.

To refine the cell gating, click on the Gate tab and adjust the dimensions of the gate. Adjust the markers as desired and apply these settings to all samples in the experiment by selecting the check mark icon, marking all the samples and selecting Accept. Run tests in triplicate and run an analysis of variance with a post hoc Bonferroni test to assess significant differences between the samples and controls.

The cell count and viability results showed that 95.2%of the cells in the sample were live and 4.8%were dead. The cell concentration was found to be 5.90 times 105 cells per milliliter. The annexin V and cell death assay showed a significant increase in apoptotic cells in SiHa cells treated with 100 nanograms per milliliter actinomycin D, compared to the controls.

The mitochondrial electrochemical transmembrane potential assay demonstrated a significant decrease in cell health profiles between actinomycin-D, medium control, and solvent control, which suggested that 100 nanograms per milliliter actinomycin D induced mitochondrial depolarization in SiHa cells. The caspase-3 and 7 assay demonstrated significant activation of terminal caspases in SiHa cells treated with 100 nanograms per milliliter actinomycin D, compared to the controls. Actinomycin D significantly induced DNA damage markers, ATM, and H2A.

X in SiHa cells, which suggested a significant increase in DNA damage. To enhance accuracy, ensure that you harvest the total cell population. Also, adhere to the manufacturer's recommended cell concentrations and staining times.

The protection of stained samples from light avoids photobleaching or fluorophores. The biochemical analysis of apoptosis by flow cytometry should be supported by microscopy. This will identify morphological features of classical apoptosis.

The features include nuclear condensation, cell membrane blending, apoptotic bodies, and karyorrhexis.