The study was financially supported by the National Research Foundation (NRF) and the South African Medical Research Council (SAMRC). We would like to acknowledge the National Health Laboratory Service (NHLS) for purchase of the Guava Muse Cell Analyzer. All figures in this publication were created with Biorender.com.

NOTE: This protocol describes steps in the cell preparation, cell enumeration, cell staining, and analysis of actinomycin D-treated SiHa cells using flow cytometric commercial assays measured and analyzed on a benchtop flow cytometer (Figure 2).
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62663/62663fig2v2.jpg" /> 
Figure 2: Workflow for the detection of biochemical apoptotic biomarkers by flow cytometry. Cells are cultured and treated as described in protocol step 1.1. (1) Cells are cultured, (2) enumerated and (3) stained with fluorescent reagents, and (4) analysed by a benchtop flow cytometer. Data are further gated, and statistically analyzed. Abbreviations: 7-AAD = 7-aminoactinomycin D; PBS = phosphate-buffered saline; PE = Phycoerythrin; TMRE = tetramethylrhodamine ethyl ester. <a href="https://www.jove.com/files/ftp_upload/62663/62663fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
1. Preparation of cell culture and treatments for flow cytometry
NOTE: Ensure aseptic technique is followed when handling cell cultures.
<ol>
	<li>Grow cell cultures in a humidified CO2 environment at 37 &#176;C with 5% CO2. Ensure that cell culture is approximately 70% confluent in the parent flask before passaging cells for experiments.</li>
	<li>Remove the medium from the flask, wash cells with 1x phosphate-buffered saline (PBS) and add 500 &#181;L of trypsin to detach the cells. After the cells begin to round and detach from the flask, sharply knock the base of the flask on the bench to detach the cells. Then neutralize the trypsin by adding approximately 5 mL of fresh culture medium supplemented with 10% fetal calf serum before counting the cells.&#160;</li>
	<li>Seed cells at 15,000 cells/mL in 3 mL of cell culture medium in a 6-well cell culture plate. Incubate the culture plates overnight at 37 &#176;C with 5% CO2 to allow the attachment of the cells to the bottom of the well.</li>
	<li>Treat the experimental cultures with 100 ng/mL actinomycin D and the medium and solvent controls with fresh culture medium and dimethyl sulfoxide (DMSO), respectively, for 24 h. Collect the spent medium in a 15 mL tube and wash cells with 1x PBS. Thereafter, add the 1x PBS wash to the tube, and then add 500 &#181;L of trypsin to the well. Next, neutralize the trypsin by adding 5 mL of fresh culture medium. 
	&#8203;NOTE: (1) Prolonged trypsin incubation or incomplete neutralization may digest the cells and compromise their cell membrane, which may skew the results. In addition, it may also change cell membrane asymmetry and, thus, the accessibility to phosphatidylserine. (2) Cells that have detached and floated to the surface can be included in the subsequent analysis by centrifuging the removed medium at 300 &#215; g for 5 min and resuspending the cells in fresh culture medium.</li>
</ol>
2. Enumerating cells using the viability assay
<ol>
	<li>Pipette 450 &#181;L of propidium iodide in a microcentrifuge tube. Add 50 &#181;L of the cell suspension to the microcentrifuge tube. Incubate the tube at room temperature for 5 min. Enumerate the cells by flow cytometry (refer to section 4).</li>
	<li>Dilute all samples to a concentration of 1 &#215; 106 cells/mL with cell culture medium before proceeding to the apoptosis assays.</li>
</ol>
3. Staining cells for flow cytometry
NOTE: Sterile conditions are not required for this part of the protocol.
<ol>
	<li>Detection of phosphatidylserine externalization using Annexin V
	<ol>
		<li>Add 100 &#181;L of cell suspension into a microcentrifuge tube. Add 100 &#181;L of a 1:1 mixture of fluorescent conjugated Annexin V and 7-AAD reagent. Incubate at room temperature for 20 min, protected from light.</li>
	</ol>
	</li>
	<li>Analysis of mitochondrial membrane depolarization
	<ol>
		<li>Centrifuge 100 &#181;L of the cell suspension at 300 &#215; g for 5 min and discard the supernatant.</li>
		<li>Resuspend the cells in 1 mL of 1x PBS, add 100 &#181;L of TMRE staining solution to each sample, and mix the suspension by gentle back-pipetting.</li>
		<li>Incubate the cells for 20 min in a humidified CO2 environment at 37 &#176;C. Wrap the samples in clean aluminum foil to protect from light. 
		NOTE: Mitochondrial membrane potential is a functional marker that is sensitive to minor changes to the cell environment. Therefore, samples should be incubated and measured under identical conditions (temperature, pH, and time elapsed between the onset of incubation and fluorescent measurement) to maintain reproducibility).&#160;Also, note that inadequate protection of samples from light causes photobleaching of fluorophores resulting in falsely low emitted fluorescence.&#160;</li>
		<li>After incubation, add 5 &#956;L of the 7-AAD staining solution to each sample and mix. Incubate for 5 min at room temperature, protected from light.</li>
	</ol>
	</li>
	<li>Detection of activated terminal caspase-3 and -7 using caspase substrate DEVD 
	NOTE: The following solutions are prepared before performing this assay.
	<ol>
		<li>Dilute the DEVD-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 at 2-8 &#176;C, protected from light. 
		NOTE: Each sample will require 5 &#181;L of this solution.</li>
		<li>Add 2 &#181;L of a 7-AAD stock solution to 148 &#181;L of 1x PBS to make the 7-AAD working solution. Store the solution on ice or at 2-8 &#176;C, protected from light. 
		NOTE: Each sample will require 150 &#181;L of this solution.</li>
		<li>Centrifuge 50 &#181;L of the cell suspension for 5 min at 300 &#215; g. Discard the supernatant. Resuspend the cells in 50 &#181;L of 1x PBS, followed by 5 &#181;L of the caspase-detection working solution. Mix thoroughly.</li>
		<li>Loosen the cap of the tubes and incubate for 30 min in a humidified CO2 environment at 37 &#176;C, protected from light. Add 150 &#181;L of the 7-AAD working solution to each sample and mix. Incubate for 5 min at room temperature, protected from light.</li>
	</ol>
	</li>
	<li>Detection of double-strand DNA breaks and total DNA damage
	<ol>
		<li>Centrifuge 50 &#181;L of the cell suspension for 5 min at 300 &#215; g. Discard the supernatant.</li>
		<li>Resuspend the cells in 500 &#181;L of 1x PBS. Add 500 &#181;L of a formaldehyde-based fixation buffer and mix. Incubate the samples on ice for 10 min.</li>
		<li>Centrifuge for 5 min at 300 &#215; g and discard the supernatant. Resuspend the cells in 90 &#181;L of 1x PBS in a microcentrifuge tube. Add 10 &#181;L of the antibody solution to the microcentrifuge tube. Incubate at room temperature for 30 min in the dark.</li>
		<li>Add 100 &#181;L of 1x PBS and centrifuge for 5 min at 300 &#215; g. Discard the supernatant. Resuspend the cells in 200 &#181;L of 1x PBS.</li>
	</ol>
	</li>
</ol>
4. Run samples on the flow cytometer.
<ol>
	<li>Verify the analytical performance of the flow cytometer by running the instrument&#39;s system check kit. Do not proceed with running samples until all checks are completed and passed.</li>
	<li>Locate the desired assay by browsing through the catalog of pre-programmed assays on the instrument and select Run Assay.
	<ol>
		<li>Mix the sample by gently back-pipetting before loading the sample onto the flow cytometer. 
		NOTE: Adequate mixing ensures cells remain in suspension and prevents low cell counts.</li>
		<li>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. Refer to the Table of Materials for instrument name and details.</li>
		<li>Using the live preview, adjust the thresholds for fluorescence and cell size and draw a rectangular gate around the cell population. Drag the threshold marker to exclude cellular debris. Select Next (Set Health Profile) to proceed. 
		NOTE: It is important to know the size of the cells. Drag the sliders and observe changes in how detected events are plotted on the real-time preview, as this will inform an accurate selection of the thresholds. If cellular debris is not excluded at this stage, it cannot be removed in post-acquisition analyses.</li>
		<li>Tap and drag the quadrant markers to separate the cell populations and so that the instrument plots detected events in real-time. Use these plots to guide the end-user on the appropriate placement of the quadrant markers. Select Next (Verify Samples) to proceed so that the instrument displays a summary of the settings. After reviewing the settings, select Next (Verify Settings) to apply these settings for all samples within the experiment. 
		NOTE: The instrument provides a 2-min live preview of cells used to adjust the instrument&#39;s settings. If this time limit expires, the instrument will release the sample, and steps 4.2.2-4.2.4 will have to be repeated. Remove the sample and mix well before reloading and continuing.</li>
		<li>Gate a population of cells by drawing a region around the cell population. Adjust thresholds for fluorescence and cell size using the sliders located on the x- and y-axis of the live preview. Apply these settings for all samples within the experiment.</li>
		<li>Mix the first sample by gently back-pipetting and load the first sample onto the flow cytometer and select Next. Name the sample and select Run so that the system begins running the sample. 
		NOTE: The instrument can only run one sample at a time.</li>
		<li>Once all samples have been run, save the experiment by giving it an appropriate title. Save the settings of the current experiment for retrieval in future runs (optional).</li>
	</ol>
	</li>
</ol>
5. Post-acquisition analysis
<ol>
	<li>If required, perform fine-tuning of gates or quadrant markers post-acquisition.</li>
	<li>Locate the experiment that needs adjustment by navigating the system&#39;s file browser and open the experiment.</li>
	<li>Tap on the thumbnail preview of the plot to enlarge it. Tap on the top-left or bottom-right corners of the cell gate to adjust the dimensions of the gate. To adjust the quadrant markers, tap on the intersection of the vertical and horizontal lines to move the markers as they are. To adjust the angle of either line, tap on the line and drag the handle.</li>
	<li>Adjust the markers (as previously described in steps 4.1.3-4.1.4) 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.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Run tests in triplicate and run an analysis of variance (ANOVA) with a post-hoc Bonferroni test to assess significant differences between the treated samples and controls. 
	NOTE: The statistical test of choice is dependent on the investigator and the variables being analyzed.</li>
</ol>

<ol>
	<li>Galluzzi, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+cell+death:+Recommendations+of+the+Nomenclature+Committee+on+Cell+Death+2018.">Molecular mechanisms of cell death: Recommendations of the Nomenclature Committee on Cell Death 2018.</a> Cell Death and Differentiation. 25 (3), 486-541 (2018).</li><li>Kerr, J. F., Wyllie, A. H., Currie, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis:+a+basic+biological+phenomenon+with+wide-ranging+implications+in+tissue+kinetics.">Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics.</a> British journal of cancer. 26 (4), 239-257 (1972).</li><li>Riccardi, C., Nicoletti, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+apoptosis+by+propidium+iodide+staining+and+flow+cytometry.">Analysis of apoptosis by propidium iodide staining and flow cytometry.</a> Nature Protocols. 1 (3), 1458-1461 (2006).</li><li>Edinger, A. L., Thompson, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Death+by+design:+apoptosis,+necrosis+and+autophagy.">Death by design: apoptosis, necrosis and autophagy.</a> Current Opinion in Cell Biology. 16 (6), 663-669 (2004).</li><li>Arends, M. J., Morris, R. G., Wyllie, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis.+The+role+of+the+endonuclease.">Apoptosis. The role of the endonuclease.</a> The American journal of pathology. 136 (3), 593-608 (1990).</li><li>Majno, G., Joris, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis,+oncosis,+and+necrosis.+An+overview+of+cell+death.">Apoptosis, oncosis, and necrosis. An overview of cell death.</a> Am J Pathol. 146 (1), 3-15 (1995).</li><li>Darzynkiewicz, Z., Traganos, F., Al-Rubeai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Apoptosis. , 33-73 (1998).</li><li>Wlodkowic, D., Skommer, J., Darzynkiewicz, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry-based+apoptosis+detection.">Flow cytometry-based apoptosis detection.</a> Methods in molecular biology. 559, 19-32 (2009).</li><li>. Introduction to Flow Cytometry Basics Available from: <a target="_blank" href="https://www.bio-rad-antibodies.com/introduction-to-flow-cytometry.html">https://www.bio-rad-antibodies.com/introduction-to-flow-cytometry.html</a> (2021)</li><li>Telford, W. G., Komoriya, A., Packard, B. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+localized+caspase+activity+in+early+apoptotic+cells+by+laser+scanning+cytometry.">Detection of localized caspase activity in early apoptotic cells by laser scanning cytometry.</a> Cytometry. 47 (2), 81-88 (2002).</li><li>Castedo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+mitochondrial+alterations+associated+with+apoptosis.">Quantitation of mitochondrial alterations associated with apoptosis.</a> J Immunol Methods. 265 (1-2), 39-47 (2002).</li><li>McKinnon, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+Cytometry:+An+Overview.">Flow Cytometry: An Overview.</a> Current protocols in immunology. 120, 1-11 (2018).</li><li>Macey, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry:+principles+and+clinical+applications.">Flow cytometry: principles and clinical applications.</a> Med Lab Sci. 45 (2), 165-173 (1988).</li><li>Darzynkiewicz, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features+of+apoptotic+cells+measured+by+flow+cytometry.">Features of apoptotic cells measured by flow cytometry.</a> Cytometry. 13 (8), 795-808 (1992).</li><li>Darzynkiewicz, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytometry+in+cell+necrobiology:+Analysis+of+apoptosis+and+accidental+cell+death+(necrosis).">Cytometry in cell necrobiology: Analysis of apoptosis and accidental cell death (necrosis).</a> Cytometry. 27 (1), 1-20 (1997).</li><li>Ormerod, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+study+of+apoptotic+cells+by+flow+cytometry.">The study of apoptotic cells by flow cytometry.</a> Leukemia. 12 (7), 1013-1025 (1998).</li><li>van Engeland, M., Nieland, L. J., Ramaekers, F. C., Schutte, B., Reutelingsperger, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+V-affinity+assay:+a+review+on+an+apoptosis+detection+system+based+on+phosphatidylserine+exposure.">Annexin V-affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure.</a> Cytometry. 31 (1), 1-9 (1998).</li><li>Vermes, I., Haanen, C., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry+of+apoptotic+cell+death.">Flow cytometry of apoptotic cell death.</a> Journal of Immunological Methods. 243 (1), 167-190 (2000).</li><li>Jahan-Tigh, R. R., Ryan, C., Obermoser, G., Schwarzenberger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytometry.">Flow cytometry.</a> J Invest Dermatol. 132 (10), 1-6 (2012).</li><li>L&#246;vborg, H., Gullbo, J., Larsson, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+apoptosis-classical+and+emerging+techniques.">Screening for apoptosis-classical and emerging techniques.</a> Anti-cancer drugs. 16 (6), 593-599 (2005).</li><li>Vorobjev, I. A., Barteneva, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-parametric+imaging+of+cell+heterogeneity+in+apoptosis+analysis.">Multi-parametric imaging of cell heterogeneity in apoptosis analysis.</a> Methods. 112, 105-123 (2017).</li><li>Telford, W. G., Komoriya, A., Packard, B. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiparametric+analysis+of+apoptosis+by+flow+and+image+cytometry.">Multiparametric analysis of apoptosis by flow and image cytometry.</a> Methods in molecular biology. 263, 141-160 (2004).</li><li>Kagami, S., Rizzo, H. L., Lee, J. J., Koguchi, Y., Blauvelt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+Th17,+Th22,+and+Th1+cells+are+increased+in+psoriasis.">Circulating Th17, Th22, and Th1 cells are increased in psoriasis.</a> J Invest Dermatol. 130 (5), 1373-1383 (2010).</li><li>. Muse&#174; Annexin V &amp; Dead Cell Kit Available from: <a target="_blank" href="https://www.luminexcorp.com/muse-annexin-v-dead-cell-kit/">https://www.luminexcorp.com/muse-annexin-v-dead-cell-kit/</a> (2019)</li><li>Roederer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+compensation+for+flow+cytometry:+visualization+artifacts,+limitations,+and+caveats.">Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats.</a> Cytometry. 45 (3), 194-205 (2001).</li><li>Kroemer, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classification+of+cell+death:+recommendations+of+the+Nomenclature+Committee+on+Cell+Death.">Classification of cell death: recommendations of the Nomenclature Committee on Cell Death.</a> Cell Death and Differentiation. 12 (2), 1463-1467 (2005).</li><li>Apoptosis assays and markers guide. Abcam Available from: <a target="_blank" href="https://www.abcam.com/kits/apoptosis-assays">https://www.abcam.com/kits/apoptosis-assays</a> (2021)</li><li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labelled+Annexin+V.">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.</a> Journal of Immunological Methods. 184 (1), 39-51 (1995).</li><li>Elmore, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis:+A+Review+of+Programmed+Cell+Death.">Apoptosis: A Review of Programmed Cell Death.</a> Toxicologic Pathology. 35 (4), 495-516 (2007).</li><li>Saelens, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toxic+proteins+released+from+mitochondria+in+cell+death.">Toxic proteins released from mitochondria in cell death.</a> Oncogene. 23 (16), 2861-2874 (2004).</li><li>Muse&#8482; MitoPotential Kit User's Guide. Luminex Corporation Available from: <a target="_blank" href="https://www.luminexcorp.com/muse-mitopotential-kit">https://www.luminexcorp.com/muse-mitopotential-kit</a> (2013)</li><li>McIlwain, D. R., Berger, T., Mak, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspase+functions+in+cell+death+and+disease.">Caspase functions in cell death and disease.</a> Cold Spring Harbor Perspectives in Biology. 5 (4), 008656 (2013).</li><li>Wigdal, S. S., Kirkland, R. A., Franklin, J. L., Haak-Frendscho, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+c+release+precedes+mitochondrial+membrane+potential+loss+in+cerebellar+granule+neuron+apoptosis:+lack+of+mitochondrial+swelling.">Cytochrome c release precedes mitochondrial membrane potential loss in cerebellar granule neuron apoptosis: lack of mitochondrial swelling.</a> Journal of Neurochemistry. 82 (5), 1029-1038 (2002).</li><li>Muse&#174; Caspase-3/7 Kit. Luminex Corporation Available from: <a target="_blank" href="https://www.luminexcorp.com/muse-caspase-3-7-kit/#overview">https://www.luminexcorp.com/muse-caspase-3-7-kit/#overview</a> (2019)</li><li>H&#228;cker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphology+of+apoptosis.">The morphology of apoptosis.</a> Cell and Tissue Research. 301 (1), 5-17 (2000).</li><li>Muse&#174; Multi-Color DNA Damage Kit User's Guide. Luminex Corporation Available from: <a target="_blank" href="https://www.luminexcorp.com/muse-multi-color-dna-damage-kit/#overview">https://www.luminexcorp.com/muse-multi-color-dna-damage-kit/#overview</a> (2020)</li><li>Kleeff, J., Kornmann, M., Sawhney, H., Korc, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actinomycin+D+induces+apoptosis+and+inhibits+growth+of+pancreatic+cancer+cells.">Actinomycin D induces apoptosis and inhibits growth of pancreatic cancer cells.</a> International journal of cancer. 86 (3), 399-407 (2000).</li><li>Wlodkowic, D., Skommer, J., Darzynkiewicz, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytometry+of+apoptosis.+Historical+perspective+and+new+advances.">Cytometry of apoptosis. Historical perspective and new advances.</a> Experimental oncology. 34 (3), 255-262 (2012).</li><li>Szeberenyi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+actinomycin+D+on+RNA+metabolism+in+human+cells.">The effect of actinomycin D on RNA metabolism in human cells.</a> Biochem Mol Biol Educ. 34 (1), 50-51 (2006).</li><li>Ginell, S., Lessinger, L., Berman, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal+and+molecular+structure+of+the+anticancer+drug+actinomycin+D--some+explanations+for+its+unusual+properties.">The crystal and molecular structure of the anticancer drug actinomycin D--some explanations for its unusual properties.</a> Biopolymers. 27 (5), 843-864 (1988).</li><li>Hou, M. H., Robinson, H., Gao, Y. G., Wang, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+actinomycin+D+bound+to+the+CTG+triplet+repeat+sequences+linked+to+neurological+diseases.">Crystal structure of actinomycin D bound to the CTG triplet repeat sequences linked to neurological diseases.</a> Nucleic Acids Res. 30 (22), 4910-4917 (2002).</li></ol>

Luminex&#174; Corporation kindly provided article processing charges.

In this study, actinomycin D-treated SiHa cells analyzed by FC revealed significant early and late biomarkers for apoptosis. Sub-optimal conditions for cell preparation, enumeration, and staining identified inaccurate results, emphasizing the need for close adherence to the manufacturer&#39;s instructions when performing FC.
This study of apoptosis by early and late biomarker identification conforms to the NCCD guidelines1 for investigating apoptosis. Actinomycin D-treated SiHa cultures demonstrated positive biomarkers for early and late apoptotic stages. The Annexin V/PI assay and the mitochondrial permeability assay showed that actinomycin D induced a PS-flip pattern and dissipation of the mitochondrial membrane transition, respectively. Once apoptotic cells reach the point of no return induced by mitochondrial perturbation, the terminal caspases are activated3,7. The activation of the terminal caspases 3 and 7 observed in this study indicates late-stage apoptosis. Furthermore, terminal caspase activation causes internucleosomal DNA cleavage and extensive DNA fragmentation, which classically has been reported as a step-ladder pattern observed by gel electrophoresis28,38.
The nuclear damage with the ATM and H2A.X FC DNA damage assay showed double-strand breaks in DNA and total DNA damage. These results confirmed classical downstream caspase-induced apoptotic nuclear damage (karyorrhexis and karyorrlysis) in experimental cultures. The use of multiple flow cytometric biomarkers thus detected the sequential multistage events in apoptosis and accurately and reproducibly identified cell populations at early and late apoptotic stages. These findings are consistent with the known pro-apoptotic features of actinomycin D in cancer treatment in humans37,39,40,41 and further support the use of actinomycin D as a positive control in FC cell culture experiments investigating apoptosis.
The gating of cell populations in this study was informed by negative medium and solvent controls, which separated apoptotic from healthy cells. Alternatively, a mixture of populations of positive and negative controls may also be used to define live and apoptotic cell populations to set cell population gates7,9. Once diseased and healthy cellular states are defined and gated, the template settings can be applied to all subsequent experimental and control cultures.
Strict adherence to the FC protocol is essential to avoid false results. During the optimization of the protocol, the following problems were observed: (1) low cell concentration, (2) high cell concentration, (3) cell clumping, (4) prolonged staining, and (5) poor sample handling. These problems can be prevented by strict adherence to optimized protocol requirements. This emphasizes the crucial nature of pre-analytical and analytical steps for FC to obtain accurate data. During cell preparation, trypsinization, pipetting, centrifugation, and dilutions must be conducted with care. Over-trypsinization and vigorous pipetting may result in chemical and mechanical shearing of cells, respectively. Prolonged and high-speed centrifugation may result in cell breakdown and high cellular debris counts. Optimal cell concentration is required to minimize the incorrect acquisition of cell events. Therefore, primary cell suspensions should be diluted to obtain optimal cell concentration.
Furthermore, while handling samples, care must be taken to prevent cell clumping and cell fragmentation and ensure that cells remain in suspension during analysis. Sample handling to prevent cell clumping allows single laminar cell flow, prevents mechanical blockage of the instrument&#39;s capillary tubing, and curbs spurious large cell size indexes. Another caveat is protecting cultures against light to avoid photo-oxidation and quenching of the fluorophores in the assays to prevent false-negative results. Care must be taken to ensure minimal light exposure at the staining step of the cells and subsequent processing steps. Moreover, prolonged immunostaining times may result in false-positive results as proteins are non-specifically stained. Therefore, adherence to manufacturer-prescribed incubation staining periods is important.
In summary, FC can accurately detect apoptosis and discriminate between early and late apoptosis biomarkers in cell culture. In addition, advances in technology have led to the manufacture of benchtop flow cytometers for non-expert scientists to study cell health and complex intracellular signaling pathways.

&#160;Apoptosis, classified as type 1 programmed cell death1, ensures an equilibrium between cell proliferation and cell death2. Apoptosis is essential during human development, post-injury, and for the prevention of diseases such as cancer3. Intrinsic and extrinsic apoptotic cell death signaling pathways4 cause sequential biochemical and morphological intracellular changes2,5,6. Morphological apoptotic features can be identified by microscopy, and the biochemical perturbation can be analyzed by biochemical assays, including flow cytometry (FC)7.
Flow cytometric analysis to identify apoptosis and understand the associated intracellular mechanisms has burgeoned over the last two decades8. FC is a scientific methodology that analyzes cells in a fluid that passes through single or multi-channel lasers (Figure 1)9,10,11. The cells in the fluid are focused into a single file by the fluidics system of the flow cytometer using hydrodynamic focusing. As cells pass through the laser, light is scattered or emitted from the cells. The scattered light can be in the forward direction (forward scatter) or toward the side (side scatter) and provides information about the cell size, and cell granularity or internal structures, respectively.
In addition, fluorescent reagents, such as fluorescent dyes or antibodies labeled with fluorophores, detect specific surface or intracellular structures or molecules. When the laser excites the fluorophores, light is emitted at a specific wavelength. Detectors&#8211;commonly photomultiplier tubes&#8211;quantify the scattered and emitted light from cell samples. The detectors produce a quantifiable current that is proportional to the light scatter and fluorescence emission. The electronic output is converted into digital signals by computing software to identify cell populations based on cell size, cell granularity, and the relative cell fluorescence of fluorophore-labeled molecules9,12,13.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62663/62663fig1v3.jpg" /> 
Figure 1: Schematic describing the technical operation and workflow of traditional flow cytometry. Cells are stained with fluorescent reagents and probed by a laser. The fluorescence signals generated are detected and converted into an electronic output, which is further digitized and analyzed by computer software and statistical programs. Abbreviations: FSC = forward scatter; SSC = side scatter. <a href="https://www.jove.com/files/ftp_upload/62663/62663fig1v3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
FC is used in both research and health diagnostics. The two goals of FC in necrobiology are the elucidation of molecular and functional properties of cell death and the discrimination of various modes of cell death14,15,16,17,18. FC applications include cell enumeration, sorting of cell populations, immunophenotyping, biomarker detection (e.g., apoptosis biomarkers), toxicity studies, and protein engineering12. In addition, FC is commonly applied to health diagnostics to assist with the diagnosis and monitoring of patients with hematological malignancies. Advances in instrumentation, fluorophore detection, and detector systems are expanding FC applications to include imaging cytometry, mass cytometry and spectral cytometry with broader research applications12.
The flow cytometric analysis of apoptosis provides advantages over traditional techniques used in assessing cell health. FC can analyze many single cells in a heterogenous sample rapidly and reproducibly to estimate apoptosis3,5. The ability of FC to provide quantitative information on cell phenotypes on an individual cell basis, and avoid bulk analysis, offers superior sensitivity to Western blotting, enzyme-linked immunosorbent assays (ELISAs), fluorometry, and spectrophotometry techniques utilized in the analysis of apoptosis8,19. Furthermore, the relative ease of FC analysis in contrast to the cumbersome and poorly reproducible manual steps of Western blots and ELISAs is advantageous. The reproducible, accurate, and high-throughput analysis of FC therefore is beneficial in cancer research20.
FC also permits the simultaneous analysis of cell cycle parameters for healthy and abnormal apoptotic cell populations21. As apoptosis is a dynamic process, different methods can produce variable results and are dependent on the time-point at which the cells are harvested22. The simultaneous quantitative assessment of multiple parameters of cell phenotype permits the detection of minor subpopulations with high accuracy, e.g., rare cell subsets with a low frequency of 0.01% can be detected23. Multi-parametric FC analysis is especially useful as apoptotic death occurs along a spectrum of early and late biochemical changes with cells at various points along the apoptotic continuum. For example, the use of dual staining using Annexin V and propidium iodide in FC analysis of apoptotic cells enables the categorization of early apoptotic cells, late apoptotic cells, and dead cells24. The accurate detection of apoptosis at multiple stages avoids misclassification and false-negative results. Thus, multiparametric analysis by FC improves the overall specificity of detecting cell phenotypes and avoids the misclassification of minor populations. Furthermore, cell sorting by FC allows the isolation of cell populations with high purity for subsequent analysis7.
The disadvantage of FC includes the use of cells in suspension, which can be challenging in tissue analysis, as disaggregation of tissue into cells may alter cellular function19. Moreover, the lack of standardization of FC instrument setup, data analysis, and assay reports&#160;may cause variation in results19, emphasizing the need for optimally training FC operators to perform, and analyze and report&#160;data. For example, the ability of FC to discriminate true apoptotic debris from apoptotic nuclei requires i) proper acquisition settings, ii) the use of calibration beads to identify a diploid DNA peak, and iii) negative and positive cell controls that are cell-specific3. Furthermore, multiparametric analysis is limited by the number of detectors, and optimal compensation needs to be performed to avoid non-specific results and spillover of fluorescent emission when using multiple fluorescent reagents25. Advances in instrument and fluorophore technology have improved parameter detection to 30 parameters12.
The identification of apoptotic cell death is not always simple7, and sensitive and specific biomarkers should be considered. The Nomenclature Committee on Cell Death (NCCD) recommends that more than one assay be used to study and quantify the process of apoptosis26. Microscopy analysis for classical apoptotic features26 is also recommended to confirm apoptosis and avoid false-positive results7. Four cardinal biochemical features that span early and late apoptotic events are (1) loss of cell membrane asymmetry; (2) dissipation mitochondrial membrane potential (&#916;&#936;m); (3) caspase activation; and (4) DNA damage26.
During early apoptosis, phosphatidylserine is externalized to the outer cell membrane27 and can be detected by Annexin V fluorescently labeled with phycoerythrin27,28,29. Furthermore, dual staining with the fluorescent DNA-binding dye, 7-aminoactinomycin D (7-AAD), distinguishes live, late-apoptotic, and dead cells. Therefore, early-apoptotic cells stain positive for Annexin V and negative for 7-AAD, in contrast to late-apoptotic cells, that stain positive for both dyes24.
Intrinsic apoptotic signals induce dissipation of the mitochondrial membrane potential (&#916;&#936;m). The disrupted &#916;&#936;m causes the release of early pro-apoptotic proteins from the mitochondrial intermembrane space into the cytosol27,29,30. Change in &#916;&#936;m can be assessed by dual staining with the positively charged, lipophilic dye, tetramethylrhodamine ethyl ester, TMRE, and 7-AAD. The TMRE dye accumulates within the inner membrane of intact mitochondria when the membrane potential is high. Depolarized mitochondria demonstrate decreased fluorescence. Live cells with polarized mitochondria (intact mitochondrial membrane) stain positive for TMRE and negative for 7-AAD. Dead cells with depolarized mitochondria stain negative for TMRE and positive for 7-AAD31.
The caspases are a family of intracellular proteases that, when activated, signal and execute apoptosis26,27. The terminal executioner caspases (3,6,7) effect late apoptosis29,32,33. Caspase-3 and -7 activities can be measured by a fluorescently labeled substrate, which, when cleaved, binds to DNA and emits a fluorescent signal. Furthermore, any compromise to cell membrane integrity can be assessed by staining with 7-AAD. Apoptotic cells stain positive for the DNA-binding dye but negative for 7-AAD. Late-apoptotic and dead cells stain positive for both dyes34.
Late apoptosis is characterized by DNA damage27,29,35, which can be evaluated by phosphorylated ataxiatelangiectasia mutated kinase (ATM) and histone H2A.X. Double-stranded DNA breaks (DSBs) cause phosphorylation of H2A.X. Fluorescently labeled antibodies against ATM and H2A.X can determine DNA damage. Negative detection of both ATM and H2A.X indicate no DNA damage, while detection of both dyes indicates the presence of double-strand breaks in the DNA36.
Actinomycin D is a potent inducer of apoptosis and acts by binding to DNA to block transcription and translation events37. This study aimed to assess biochemical apoptosis induced by actinomycin D in the SiHa cell line by analyzing early- and late-stage biomarkers of apoptosis. Four biochemical biomarkers of apoptosis assessed the sequential steps in the apoptotic cascade that included loss of cell membrane asymmetry, change in mitochondrial membrane potential, activation of terminal caspases, and DNA damage.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6-well plates</td>
			<td>Lasec</td>
			<td>P1PLA044C-000006</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher</td>
			<td>41966052</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Sigma-Aldrich</td>
			<td>P10-040500</td>
			<td></td>
		</tr>
		<tr>
			<td>Guava Muse Cell Analyzer</td>
			<td>Luminex</td>
			<td>0500-3115</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes/Eppendorf</td>
			<td>Merck</td>
			<td>EP0030122208-200EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Muse Annexin V kit</td>
			<td>Merck</td>
			<td>MCH100105</td>
			<td></td>
		</tr>
		<tr>
			<td>Muse Caspase-3/7 kit</td>
			<td>Merck</td>
			<td>MCH100108</td>
			<td></td>
		</tr>
		<tr>
			<td>Muse Count and Viability kit</td>
			<td>Merck</td>
			<td>MCH600103</td>
			<td></td>
		</tr>
		<tr>
			<td>Muse DNA Damage kit</td>
			<td>Merck</td>
			<td>MCH200107</td>
			<td></td>
		</tr>
		<tr>
			<td>Muse MitoPotential kit</td>
			<td>Merck</td>
			<td>MCH100110</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS Buffer</td>
			<td>ThermoFisher</td>
			<td>70013065</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-strep</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>SiHa cells</td>
			<td>ATCC</td>
			<td>CRL-1550</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 culture flasks</td>
			<td>Sigma-Aldrich</td>
			<td>C6231</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Pan Biotech</td>
			<td>P10-040500</td>
			<td></td>
		</tr>
	</tbody>
</table>

flow cytometric analysis of apoptotic biomarkers in actinomycin d-treated siha cervical cancer cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 &#176;C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.

The cell count and viability (Figure 3) results showed that 95.2% of the cells in the sample were live, and 4.8% were dead. The total cell concentration in the original sample was 6.20 x 106 cells/mL.
The Annexin V and cell death assay (Figure 4) showed a significant increase (p &#60; 0.0001) in apoptotic cells in SiHa cells treated with 100 ng/mL actinomycin D in comparison to the controls. Because Annexin V staining is increased in cells during early stage apoptosis, this finding suggests that 100 ng/mL actinomycin D induced apoptosis in SiHa cells.
The mitochondrial electrochemical transmembrane potential assay (Figure 5) demonstrated a significant decrease (p &#60; 0.0001) in cell health profiles (live, depolarized and live, depolarized, and dead, dead) between actinomycin D, medium control, and solvent control. These data suggest that 100 ng/mL actinomycin D induced mitochondrial depolarization in SiHa cells.
The caspase 3/7 assay (Figure 6) demonstrated significant (p &#60; 0.0001) activation of caspases 3 and 7 in SiHa cells treated with 100 ng/mL actinomycin D compared to the controls. These finding demonstrates that 100 ng/mL actinomycin D induced apoptosis in SiHa cells.
The DNA damage assay (Figure 7) showed that 100 ng/mL actinomycin D significantly (p &#60; 0.0001) induced DNA damage markers, ATM and H2A.X, in SiHa cells. This finding suggests&#160;significant increase in DNA damage in SiHa cells treated with actinomycin D.
The results of suboptimal experiments (Figure 8) demonstrate analytical considerations across all assays. Cell concentration affects accuracy of data. In Figure 8A, the cell count is unacceptably low. The gated cell populations in all 4 quadrants of the dot plot have low signal intensity. The manufacturer optimizes&#160;the assay for 300-700 cells/&#181;L in the final sample volume.&#160;This example illustrates the importance of using the correct sample concentration prescribed by the manufacturer.
In addition, high cell concentrations also caused erroneous results (Figure 8B). Medium and experimental cultures demonstrated 99.95% and 100% live cells, respectively. The flow rate for both assays exceeded the manufacturer&#39;s optimized concentration of 100-500 cells/&#956;L and required dilution with 1x Assay Buffer to avoid inaccurate analysis.&#160;
Cell clumping should be avoided during the preparation of experimental cultures as it produces false results due to increased cell size indices, as illustrated in the Annexin V assay. Figure 8C shows a dual problem of high cell concentration that exceeds the manufacturer&#39;s instruction and cell clumping, as evidenced by cell size indices exceeding 4 in the SiHa medium control. The high cell concentration is illustrated by the sheeting of cells forming a bright red plane of cells in medium cultures, demonstrating discordantly high apoptotic cell populations.
Prolonged staining of cultures can result in non-specific binding of proteins and result in false results, as demonstrated by the Annexin V assay. Figure 8D demonstrates similar results for medium and experimental cultures due to prolonged staining.&#160;
Poor sample handling, extended trypsinization of adherent cells,&#160;vigorous pipetting during wash steps, and high-speed and prolonged centrifugation steps cause cell lysis and high amounts of cell debris. In Figure 8E, cultures analyzed by the caspase 3/7 assay demonstrate increased cell debris evidenced by a small cell size index (&#60;2.2 cell size index). Care should therefore be taken when preparing samples for data acquisition.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62663/62663fig03.jpg" /> 
Figure 3: Cell Count and viability assay. The population profile separates debris from live and dead cells. The two-dimensional dot plot is gated and divides live and dead cell populations. The information panel provides quantitative data on cell count, percentage of total viable cells, and the total number of viable cells in the sample. These data can be used to standardize the cell count in all samples for subsequent apoptosis analyses. <a href="https://www.jove.com/files/ftp_upload/62663/62663fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62663/62663fig04.jpg" /> 
Figure 4: Annexin V assay. All samples show identical gating parameters, and statistical analysis of each sub-population reveals a significant increase in apoptosis in cells treated with actinomycin D. All assays were performed as three independent experiments, and each experiment was assayed in triplicate. Data are presented as mean &#177; SD, and p &#60; 0.05 was considered statistically significant. **** p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/62663/62663fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62663/62663fig05.jpg" /> 
Figure 5: Mitochondrial electrochemical transmembrane potential assay. All samples show identical gating parameters, and statistical analysis of each sub-population reveals significant perturbation in mitochondrial membrane potential in cells treated with actinomycin D. All assays were performed as three independent experiments, and each experiment was assayed in triplicate. Data are presented as mean &#177; SD, and p &#60; 0.05 was considered statistically significant. **** p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/62663/62663fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62663/62663fig06.jpg" /> 
Figure 6: Caspase 3/7 detection assay. All samples show identical gating parameters, and statistical analysis of each sub-population reveals a significant increase in caspase 3/7 activity in cells treated with actinomycin D. All assays were performed as three independent experiments, and each experiment was assayed in triplicate. Data are presented as mean &#177; SD, and p &#60; 0.05 was considered statistically significant. **** p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/62663/62663fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/62663/62663fig07.jpg" /> 
Figure 7: DNA damage assay. All samples show identical gating parameters, and statistical analysis of each sub-population reveals a significant increase in double-strand DNA damage and total DNA damage in cells treated with actinomycin D. All assays were performed on three independent experiments, and each experiment was assayed in triplicate. Data are presented as mean &#177; SD, and p &#60; 0.05 was considered statistically significant. * p &#60; 0.05; *** p &#60; 0.001; **** p &#60; 0.0001. <a href="https://www.jove.com/files/ftp_upload/62663/62663fig07large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/62663/62663fig8v2.jpg" /> 
Figure 8: Sub-optimal experimental conditions yield poor results. (A) Low concentration of cells, (B) high concentration of cells, (C) cell clumping and aggregation evident by high cell size index, (D) prolonged staining of cell samples evident by increased positive staining in both samples, and (E) poor sample handling evident by increased cellular debris (cell size index &#60; 2.2). <a href="https://www.jove.com/files/ftp_upload/62663/62663fig8v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Flow Cytometric Analysis of Apoptotic Biomarkers in Actinomycin D-Treated SiHa Cervical Cancer Cells at JoVE.com

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

Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer.

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V ...

flow-cytometric-analysis-apoptotic-biomarkers-actinomycin-d-treated

Research

JoVE Journal

Cancer Research

4.1K Views.  University of Pretoria. Apoptosis can be characterized by flow cytometric analysis of early and late apoptotic biomarkers. The cervical cancer cell line, SiHa, was analyzed for apoptosis biomarkers after treatment with Actinomycin D using a benchtop flow cytometer. 

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

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

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 named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.

Here, we present a protocol to isolate apoptotic breast cancer cells by fluorescence-activated cell sorting and further detect the transition of breast non-stem cancer cells to breast cancer stem cell-like cells after apoptosis reversal by flow cytometry.

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

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Radiosensitivity of Cancer Stem Cells in Lung Cancer Cell Lines

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide clues to overcoming radioresistance. Voltage-gated calcium channel &#945;2&#948;1 subunit isoform 5 has been reported as a marker for radioresistant CSCs in non-small cell lung cancer (NSCLC) cell lines. Using calcium channel &#945;2&#948;1 subunit as an example of a CSC marker, methods to study the radiosensitivity of CSCs in NSCLC cell lines are presented. CSCs are sorted with putative markers by flow cytometry, and the self-renewal capacity of sorted cells is evaluated by sphere formation assay. Colony formation assay, which determines how many cells lose the ability to generate descendants forming the colony after a certain dose of radiation, is then performed to assess the radiosensitivity of sorted cells. This manuscript provides initial steps for studying the radiosensitivity of CSCs, which establishes the basis for further understanding of the underlying mechanisms.

The presence of cancer stem cells have been associated with relapse or poor outcomes after radiotherapy. This manuscript describes the methods to study the radiosensitivity of cancer stem cells in lung cancer cell lines.

The presence of cancer stem cells (CSCs) has been associated with relapse or poor outcomes after radiotherapy. Studying radioresistant CSCs may provide ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Two Flow Cytometric Approaches of NKG2D Ligand Surface Detection to Distinguish Stem Cells from Bulk Subpopulations in Acute Myeloid Leukemia

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties (so called leukemic stem cells, LSCs) from more differentiated counterpart leukemic cells that lack disease initiation potential although they carry similar leukemia specific genetic mutations. NKG2DL are biochemically highly diverse MHC class I-like self-molecules. Healthy cells in homeostatic conditions generally do not express NKG2DL on the cell surface. Instead, expression of these ligands is induced upon exposure to cellular stress (e.g., oncogenic transformation or infectious stimuli) to trigger elimination of damaged cells via lysis through NKG2D-receptor-expressing immune cells such as natural killer (NK) cells. Interestingly, NKG2DL surface expression is selectively suppressed in LSC subpopulations, allowing these cells to evade NKG2D-mediated immune surveillance. Here, we present a side-by-side analysis of two different flow cytometry methods that allow the investigation of NKG2DL surface expression on cancer cells i.e., a method involving pan-ligand recognition and a method involving staining with multiple antibodies against single ligands. These methods can be used to separate viable NKG2DL negative cellular subpopulations with putative cancer stem cell properties from NKG2DL positive non-LSC.

We present two different staining protocols for NKG2D ligand (NKG2DL) detection in human primary acute myeloid leukemia (AML) samples. The first approach is based on a fusion protein, able to recognize all known and potentially yet unknown ligands, while the second protocol relies on the addition of multiple anti-NKG2DL antibodies.

Within the same patient, absence of NKG2D ligands (NKG2DL) surface expression was shown to distinguish leukemic subpopulations with stem cell properties ...

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated molecular patterns and inducing the production of type I interferon. Immunotherapies, including checkpoint inhibition, primarily rely on preexisting tumor-specific T cells to unfold a therapeutic effect. Thus, synergistic therapeutic approaches that exploit immunogenic cell death as an intrinsic anti-cancer vaccine may improve their responsiveness. However, the spectrum of immunogenic factors released by cells under therapy-induced stress remains incompletely characterized, especially regarding extracellular vesicles (EVs). EVs, nano-scale membranous particles emitted from virtually all cells, are considered to facilitate intercellular communication and, in cancer, have been shown to mediate cross-priming against tumor antigens. To assess the immunogenic effect of EVs derived from tumors under various conditions, adaptable, scalable, and valid methods are sought-for. Therefore, herein a relatively easy and robust approach is presented to assess EVs' in vivo immunogenicity. The protocol is based on flow cytometry analysis of splenic T cells after in vivo immunization of mice with EVs, isolated by precipitation-based assays from tumor cell cultures under therapy or steady-state conditions. For example, this work shows that oxaliplatin exposure of B16-OVA murine melanoma cells resulted in the release of immunogenic EVs that can mediate the activation of tumor-reactive cytotoxic T cells. Hence, screening of EVs via in vivo immunization and flow cytometry identifies conditions under which immunogenic EVs can emerge. Identifying conditions of immunogenic EV release provides an essential prerequisite to testing EVs' therapeutic efficacy against cancer and exploring the underlying molecular mechanisms to ultimately unveil new insights into EVs' role in cancer immunology.

In Vivo Immunogenicity Screening of Tumor-Derived Extracellular Vesicles by Flow Cytometry of Splenic T Cells

This manuscript describes how to assess in vivo immunogenicity of tumor cell-derived extracellular vesicles (EVs) using flow cytometry. EVs derived from tumors undergoing treatment-induced immunogenic cell death seem particularly relevant in tumor immunosurveillance. This protocol exemplifies the assessment of oxaliplatin-induced immunostimulatory tumor EVs but can be adapted to various settings.

Immunogenic cell death of tumors, caused by chemotherapy or irradiation, can trigger tumor-specific T cell responses by releasing danger-associated ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...