This study was supported by a Natural Sciences and Engineering Council of Canada (NSERC) research grant and an Alberta Agriculture Funding Consortium grant to DRB. AMR is supported through an NSERC Vanier Canadian Graduate Scholarship, a University of Alberta teaching assistantship and a Queen Elizabeth II graduate scholarship.

This procedure can be performed in 500 μL siliconized tubes or 6 mL polystyrene round-bottom FACS tubes. The latter is normally used when carrying out viability analysis in conjunction with other procedures. All volumes given are for 6 mL polystyrene round-bottom FACS tubes. For 500 μL siliconized tubes, reduce all volumes by 1/5.
The optimum concentration for flow cytometry analysis is 2-4 x 106 cells per 200 μL volume. Cell loss may result from this procedure and thus we recommend that each sample consist of 4 x 106 cells at the start of the procedure.
1. Cell Preparation
<ol>
	<li>Harvest cells - refer to specific procedures for corresponding cell lines or primary cell isolations.</li>
	<li>Centrifuge samples at 335 x g for 10 minutes and decant the supernatant.</li>
	<li>Resuspend cells in 2 mL 1 x phosphate buffered saline (PBS) -/- (no calcium, no magnesium).</li>
	<li>Centrifuge samples at 335 x g for 10 minutes and decant the supernatant.</li>
	<li>Resuspend cells in 1 mL 1 x Annexin V binding buffer.</li>
	<li>Centrifuge samples at 335 x g for 10 minutes and decant the supernatant.</li>
	<li>Resuspend cells in 100 μL 1 x Annexin V binding buffer.</li>
</ol>
2. Application of Annexin V/ PI Stain
<ol>
	<li>Add Annexin V according to the manufacturer's recommendations [e.g. 5 μL Annexin V Alexa Fluor 488 (Molecular Probes, A13201)].</li>
	<li>Incubate tubes in the dark for 15 minutes at room temperature.</li>
	<li>Add 100 μL of 1 x Annexin V binding buffer to each reaction tube. There should be approximately 200 μL in each tube.</li>
	<li>Add 4 μL of PI (Sigma, Cat# P-4864-10ML) that has been diluted 1:10 in 1 x Annexin V binding buffer (i.e. 1 μL PI with 9 μL 1 x Annexin V binding buffer). This will yield a final PI concentration of 2 μg/mL in each sample.</li>
	<li>Incubate tubes in the dark for 15 minutes at room temperature.</li>
	<li>Add 500 μL 1 x Annexin V binding buffer to wash the cells.</li>
	<li>Centrifuge samples at 335 x g for 10 minutes and decant the supernatant.</li>
	<li>Resuspend cells in 500 μL 1 x Annexin V binding buffer and 500 μL 2% formaldehyde to create a 1% formaldehyde (fixative) solution. Mix tubes by gentle flicking.</li>
	<li>Fix samples on ice for 10 minutes. Alternatively, samples can be stored overnight at 4°C in the dark. If the latter method is chosen, make sure to be consistent across all tubes labeled with Annexin V/PI (including compensation controls).</li>
	<li>Add 1 mL 1 x PBS-/- to each sample and mix gently by flicking.</li>
	<li>Centrifuge tubes at 425 x g for eight minutes and decant the supernatant.</li>
	<li>Repeat steps 2.10 and 2.11.</li>
	<li>Resuspend pellet by flicking the tube.</li>
	<li>Add 16 μL of 1:100 diluted RNase A (Sigma, R4642) to give a final concentration of 50 μg/mL. Incubate for 15 min at 37°C.</li>
	<li>Add 1 mL 1 x PBS-/- and mix gently by flicking.</li>
	<li>Centrifuge tubes at 425 x g for eight minutes.</li>
	<li>Samples are now ready to be analyzed. Alternatively, samples can be used for subsequent staining steps if Annexin/PI staining is being performed in parallel with other procedures.</li>
</ol>
3. Representative Results:
Examples of cell types and cell lines that have varying degrees of false-positive PI staining are shown in Figure 1. To account for the false-positive events, cells were fixed and then treated with RNase A. This results in a significant decrease in the number of false-positive events, compared to cells that didn't undergo RNase treatment (Figure 2B). Figure 2C shows representative images of cells that underwent RNase treatment, compared to cells that had no RNase treatment. Figure 3 shows how the modified Annexin V/PI protocol, with fixation and RNase treatment steps, improves upon conventional protocols by limiting the number of false-positive staining events.
<img src="/files/ftp_upload/2597/2597fig1.jpg" alt="Fluorescence microscopy diagram comparing false and true positives in cell staining using PI/DRAQ5." data-altupdatedat="1747911167000" data-alt="Figure 1"> 
Figure 1. Conventional propidium iodide staining protocols lead to a significant number of false positive events. We have previously evaluated extent of false positive staining across primary cells across a broad range of animal models as well as in a variety of cell lines 10. Representative images show extent of false positive staining in three unique cell populations (one commonly used cell line - RAW macrophages and two primary cells - murine bone marrow macrophages (BMM) and goldfish primary kidney macrophages (PKM)). True positive events display the expected co-localization between PI and DRAQ5 within the nuclear compartment (yellow areas depict colocalization between PI and DRAQ5). DRAQ5 is highly membrane permeable dye that accurately stains nuclear compartment in both live and dead cells 10,11,12. For easier visual determination of colocalization DRAQ5 stain was given a green pseudocolour.
<img src="/files/ftp_upload/2597/2597fig2.jpg" alt="Cell staining comparison via bar graphs and fluorescence microscopy, analyzing DNA content and RNase effect." data-altupdatedat="1747911167000" data-alt="Figure 2"> 
Figure 2. Improving the accuracy of PI nuclear staining. (A) We assessed accuracy of PI nuclear staining based on degree of similarity of a number of well-characterized nuclear stains (BrdU, 7-AAD and DAPI) to DRAQ5. BrdU is a synthetic nucleoside that is incorporated into the DNA of proliferating cells, and as such will only stain within the nuclear compartment. 7-AAD has a high affinity for DNA and, similar to PI, cannot cross an intact plasma membrane. DAPI also has a strong affinity for DNA and is the most commonly used nuclear stain in fluorescent microscopy. The degree of similarity was &gt;99% between BrdU, 7-AAD, DAPI and DRAQ5, highlighting their ability to accurately stain the cell nucleus in RAW 264.7 macrophages. In contrast, cytoplasmic PI staining based on conventional protocols leads to a marked decrease in percent similarity of staining relative to DRAQ5. (B) Similar results are observed in two primary cells tested: murine bone marrow macrophages (BMM) and goldfish primary kidney macrophages (PKM). Incorporation of 50 μg/mL RNase A at specific stage within the staining procedure removes non-nuclear PI staining and significantly increases degree of similarity relative to DRAQ5 staining. (C) Representative images of primary kidney macrophages show the degree of similarity for cells with and without RNase treatment. For easier visual determination of differences between treatments, DRAQ5 was given a green. Yellow areas depict colocalization between BrdU and DRAQ5, and PI and DRAQ5.
<img src="/files/ftp_upload/2597/2597fig3.jpg" alt="Flow cytometry scatter plot and bar graph: cell viability with and without RNase treatment." data-altupdatedat="1747911167000" data-alt="Figure 3"> 
Figure 3. Modified Annexin V/PI significantly reduces false apoptosis/necrotic events. Primary goldfish kidney macrophages (PKM) were stained with conventional and modified Annexin V/PI protocols. Scatterplots depict the Annexin V/PI stain in goldfish PKM. The scatterplot on the left shows conventional Annexin V/PI staining (no RNase) of PKM cells. The scatterplot on the right shows PKM cells stained with the modified Annexin V/PI protocol (with RNase). The addition of RNase results in a marked reduction of PI staining due to the removal of false positive stains. PKM cells were then analyzed based on distinct populations within the cultures- early progenitors (EP), monocytes (Mono) and mature macrophages (Mat mac). The reduction in the number of positive PI events, due to removal of false positive events is more pronounced in large mature macrophage cells than in smaller early progenitor cells. Conclusions based on conventional protocols would have erroneously suggested a positive correlation in cellular death for more mature macrophage subsets.

<ol><li>Vermes, I., Haanen, C., Steffens-Nakken, H., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+assay+for+apoptosis.+Flow+cytometric+detection+of+phosphatidylserine+expression+on+early+apoptotic+cells+using+fluorescein+labeled+Annexin+V%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled Annexin V.</a> J Immunol Methods. 184, 39-51 (1995).</li>
<li>Vermes, I., Haanen, C., Reutelingsperger, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytometry+of+apoptotic+cell+death%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Flow cytometry of apoptotic cell death.</a> J Immunol Methods. 243, 167-190 (2000).</li>
<li>Cornelissen, M., Philippe, J., De Sitter, S., De Ridder, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Annexin+V+expression+in+apoptotic+peripheral+blood+lymphocytes%3A+An+electron+microscopic+evaluation%5BTitle%5D)+AND+%22Apoptosis%22%5BJournal%5D)">Annexin V expression in apoptotic peripheral blood lymphocytes: An electron microscopic evaluation.</a> Apoptosis. 7, 41-47 (2002).</li>
<li>Fried, J., Perez, A. G., Clarkson, B. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Flow+cytofluorometric+analysis+of+cell+cycle+distributions+using+propidium+iodide.+Properties+of+the+method+and+mathematical+analysis+of+the+data%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Flow cytofluorometric analysis of cell cycle distributions using propidium iodide. Properties of the method and mathematical analysis of the data.</a> J Cell Biol. 71, 172-181 (1976).</li>
<li>Bacso, Z., Everson, R. B., Eliason, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+DNA+of+Annexin+V-binding+apoptotic+cells+is+highly+fragmented%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">The DNA of Annexin V-binding apoptotic cells is highly fragmented.</a> Cancer Res. 60, 4623-4628 (2000).</li>
<li>Darzynkiewicz, Z., Bruno, S., Del Bino, G., Gorczyca, W., Hotz, M. A., Lassota, P., Traganos, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Features+of+apoptotic+cells+measured+by+flow+cytometry%5BTitle%5D)+AND+%22Cytometry%22%5BJournal%5D)">Features of apoptotic cells measured by flow cytometry.</a> Cytometry. 13, 795-808 (1992).</li>
<li>Kroemer, G., Dallaporta, B., Resche-Rigon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mitochondrial+death%2Flife+regulator+in+apoptosis+and+necrosis%5BTitle%5D)+AND+%22Annu.+Rev.+Physiol%22%5BJournal%5D)">The mitochondrial death/life regulator in apoptosis and necrosis.</a> Annu. Rev. Physiol. 60, 619-642 (1998).</li>
<li>Denecker, G., Vercammen, D., Declercq, W., Vandenabeele, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Apoptotic+and+necrotic+cell+death+induced+by+death+domain+receptors%5BTitle%5D)+AND+%22Cell+Mol.+Life+Sci%22%5BJournal%5D)">Apoptotic and necrotic cell death induced by death domain receptors.</a> Cell Mol. Life Sci. 58, 356-370 (2001).</li>
<li>Faleiro, L., Lazebnik, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Caspases+disrupt+the+nuclear-cytoplasmic+barrier%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Caspases disrupt the nuclear-cytoplasmic barrier.</a> J Cell Biol. 151, 951-959 (2000).</li>
<li>Rieger, A. M., Hall, B. E., Luong, L. T., Schang, L. M., Barreda, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conventional+apoptosis+assays+using+propidium+iodide+generate+a+significant+number+of+false+positives+that+prevent+accurate+assessment+of+cell+death%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">Conventional apoptosis assays using propidium iodide generate a significant number of false positives that prevent accurate assessment of cell death.</a> J Immunol Methods. 358, 81-92 (2010).</li>
<li>Edward, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Minireview%3A+Use+of+DNA-specific+anthraquinone+dyes+to+directly+reveal+cytoplasmic+and+nuclear+boundaries+in+live+and+fixed+cells%5BTitle%5D)+AND+%22Mol.+Cells%22%5BJournal%5D)">Minireview: Use of DNA-specific anthraquinone dyes to directly reveal cytoplasmic and nuclear boundaries in live and fixed cells.</a> Mol. Cells. 27, 391-396 (2009).</li>
<li>Njoh, K. L., Patterson, L. H., Zloh, M., Wiltshire, M., Fisher, J., Chappell, S., Ameer-Beg, S., Bai, Y., Matthews, D., Errington, R. J., Smith, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spectral+analysis+of+the+DNA+targeting+bisalkylaminoanthraquinone+DRAQ5+in+intact+living+cells%5BTitle%5D)+AND+%22Cytometry+Part+A%22%5BJournal%5D)">Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells.</a> Cytometry Part A. 69, 805-814 (2006).</li>
</ol>

No conflicts of interest declared.

The Annexin V/PI protocol presented here is a modified version of conventional protocols and takes into account the presence of RNA in the cytoplasm which also has high affinity for PI. Introduction of RNase A (50 &mu;g/mL) following a 1% formaldehyde fixation step late in the staining procedure significantly improves accuracy of nuclear PI staining. No negative effects are observed in nuclear PI staining or plasma membrane Annexin V staining. Without RNase A treatment, up to 40% of PI stain results can lead to false positive events, which leads to a potentially significant and negative impact on downstream conclusions10.
Our modified Annexin V/PI staining protocol is simple and has been used effectively in a broad range of cell types (Primary cells: murine bone marrow macrophages and splenocytes; swine lung resident cells, lung alveolar macrophages, mesenteric lymph node isolates, peripheral blood leukocytes, splenocytes; chicken blastoderm cells; goldfish primary kidney macrophages; carp peripheral blood leukocytes; Cell lines: RAW 264.7 macrophages, Jurkat T cells, swine 3D4/31 macrophages, CCL-71 goldfish fin fibroblasts, 3B11 catfish B cells 10). It is important to note that cells are differentially affected by false positive PI staining, with primary cells generally having a greater number of false positive events 10. The differences false positive PI staining among these cellular populations may be partially attributed to differences in cell size, but may also result from differences in RNA content. As such, incorporation of this procedure is particularly relevant for experimental systems that utilize, among others, cells undergoing genotoxic stress, cells treated with cell cycle arrest drugs such as thymidine or hydroxyurea, virally-infected cells, and studies on embryonic cells where developmental progression is characterized by discrete changes in cellular RNA synthesis.

<table><tbody><tr><td>1 x PBS-/-</td><td></td><td></td><td></td></tr><tr><td>1 x Annexin V Binding Buffer</td><td>BD Biosciences</td><td>556454</td><td></td></tr><tr><td>Annexin V-Alexa Fluor 488</td><td>Molecular Probes, Life Technologies</td><td>A13201</td><td></td></tr><tr><td>Propidium iodide (PI)</td><td>Sigma-Aldrich</td><td>P4864</td><td>1 mg/mL in H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>2% Formaldehyde</td><td>Sigma-Aldrich</td><td>F1268</td><td></td></tr><tr><td>RNase A from bovine pancreas</td><td>Sigma-Aldrich</td><td>R4642</td><td></td></tr><tr><td>DRAQ5</td><td>Biostatus</td><td>DR50050</td><td>Dilute 1:60 in 1 x PBS-/-</td></tr></tbody></table>

modified annexin v/propidium iodide apoptosis assay for accurate assessment of cell death

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely used in conjunction with Annexin V to determine if cells are viable, apoptotic, or necrotic through differences in plasma membrane integrity and permeability1,2. The Annexin V/ PI protocol is a commonly used approach for studying apoptotic cells3. PI is used more often than other nuclear stains because it is economical, stable and a good indicator of cell viability, based on its capacity to exclude dye in living cells 4,5. The ability of PI to enter a cell is dependent upon the permeability of the membrane; PI does not stain live or early apoptotic cells due to the presence of an intact plasma membrane 1,2,6. In late apoptotic and necrotic cells, the integrity of the plasma and nuclear membranes decreases7,8, allowing PI to pass through the membranes, intercalate into nucleic acids, and display red fluorescence 1,2,9. Unfortunately, we find that conventional Annexin V/ PI protocols lead to a significant number of false positive events (up to 40%), which are associated with PI staining of RNA within the cytoplasmic compartment10. Primary cells and cell lines in a broad range of animal models are affected, with large cells (nuclear: cytoplasmic ratios &lt;0.5) showing the highest occurrence10. Herein, we demonstrate a modified Annexin V/ PI method that provides a significant improvement for assessment of cell death compared to conventional methods. This protocol takes advantage of changes in cellular permeability during cell fixing to promote entry of RNase A into cells following staining. Both the timing and concentration of RNase A have been optimized for removal of cytoplasmic RNA. The result is a significant improvement over conventional Annexin V/ PI protocols (&lt; 5% events with cytoplasmic PI staining).

Watch this Scientific Journal Video about Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death at JoVE.com

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

An accurate method for the assessment of cell death is described. The protocol improves upon conventional Annexin V/ propidium iodide (PI) protocols, which display up to 40% false- positive events in cell lines and primary cells from a broad range of animal models.

Studies of cellular apoptosis have been significantly impacted since the introduction of flow cytometry-based methods. Propidium iodide (PI) is widely ...

modified-annexin-vpropidium-iodide-apoptosis-assay-for-accurate

Research

JoVE Journal

Biology

103.2K Views.  University of Alberta. Een nauwkeurige methode voor de beoordeling van de cel dood is beschreven. Het protocol verbetert conventionele Annexine V / propidiumjodide (PI) protocollen, die up display tot 40% vals-positieve gebeurtenissen in cellijnen en primaire cellen van een breed scala van diermodellen.

Video: Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

Detection and Isolation of Apoptotic Bodies to High Purity

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. It has been proposed that ApoBDs can aid cell clearance as well as intercellular communication through trafficking biomolecules. Conventional approaches used for the identification and isolation of ApoBDs are often limited by the lack of accurate quantification and low sample purity. Here, we describe a workflow to confirm the induction of apoptosis, validate ApoBD formation, and isolate ApoBDs to high purity. We will also outline and compare fluorescence-activated cell sorting (FACS) and differential centrifugation based approaches to isolate ApoBDs. Furthermore, the purity of isolated ApoBDs will be confirmed using a previously establish flow cytometry-based staining and analytical method. Taken together, using the described approach, THP-1 monocyte apoptosis and apoptotic cell disassembly was induced and validated, and ApoBD generated from THP-1 monocytes were isolated to a purity of 97-99%.

A workflow using flow cytometry or differential centrifugation is developed to detect, quantify and isolate apoptotic bodies from an apoptotic sample to high purity.

Apoptotic bodies (ApoBDs), microvesicles and exosomes are the key members of the extracellular vesicle family, with ApoBDs being one of the largest type. ...

Biochemistry

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depolarization

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a reduction in neuronal population and function. Damaged axons can release excitatory molecules including potassium or glutamate into the extracellular matrix, which in turn, can produce further insult and injury to the supporting glial cells including astrocytes and oligodendrocytes 8, 16. If the insult persists, cells will undergo programmed cell death (apoptosis), which is regulated and activated by a number of well-established signal transduction cascades 14. Apoptosis and tissue necrosis can occur after traumatic brain injury, cerebral ischemia, and seizures. A classical example of apoptotic regulation is the family of cysteine-dependent aspartate-directed proteases, or caspases. Activated proteases including caspases have also been implicated in cell death in response to chronic neurodegenerative diseases including Alzheimer's, Huntington's, and Multiple Sclerosis 4, 14, 3, 11, 7.
In this protocol we describe the use of the NucView 488 caspase-3 substrate to measure the rate of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte (OLG) cell cultures 15, 5, following exposure to different extracellular stresses such as high concentrations of potassium or glutamate. The conditionally-immortalized N19-OLG cell line (representing the O2A progenitor) was obtained from Dr. Anthony Campagnoni (UCLA Semel Institute for Neuroscience) 15, 5, and has been previously used to study molecular mechanisms of myelin gene expression and signal transduction leading to OLG differentiation (e.g.6, 10). We have found this cell line to be robust with respect to transfection with exogenous myelin basic protein (MBP) constructs fused to either RFP or GFP (red or green fluorescent protein) 13, 12. Here, the N19-OLG cell cultures were treated with either 80 mM potassium chloride or 100 mM sodium glutamate to mimic axonal leakage into the extracellular matrix to induce apoptosis 9. We used a bi-functional caspase-3 substrate containing a DEVD (Asp-Glu-Val-Asp) caspase-3 recognition subunit and a DNA-binding dye 2. The substrate quickly enters the cytoplasm where it is cleaved by intracellular caspase-3. The dye, NucView 488 is released and enters the cell nucleus where it binds DNA and fluoresces green at 488 nm, signaling apoptosis. Use of the NucView 488 caspase-3 substrate allows for live-cell imaging in real-time 1, 10. In this video, we also describe the culturing and transfection of immortalized N19-OLG cells, as well as live-cell imaging techniques.

Monitoring Cleaved Caspase-3 Activity and Apoptosis of Immortalized Oligodendroglial Cells using Live-cell Imaging and Cleaveable Fluorogenic-dye Substrates Following Potassium-induced Membrane Depola

Live-cell imaging of caspase-3 mediated apoptosis in immortalized N19-oligodendrocyte cell cultures using the NucView 488 caspase-3 substrate. This technique is applicable for programmed cell death assays in real-time in a variety of cell types and tissues.

The central nervous system can experience a number of stresses and neurological insults, which can have numerous adverse effects that ultimately lead to a ...

Neuroscience

An Automated Differential Nuclear Staining Assay for Accurate Determination of Mitocan Cytotoxicity

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes more complex, the goal of targeting mitochondria using various compounds that inflict mitochondrial damage (so-called mitocans) is becoming quite popular. Unfortunately, many existing cytotoxicity assays, such as those based on tetrazolium salts or resazurin require functional mitochondrial enzymes for their performance. The damage inflicted by compounds that target mitochondria often compromises the accuracy of these assays. Here, we describe a modified protocol based on differential staining with two fluorescent dyes, one of which is cell-permeant (Hoechst 33342) and the other of which is not (propidium iodide). The difference in staining allows living and dead cells to be discriminated. The assay is amenable to automated microscopy and image analysis, which increases throughput and reduces bias. This also allows the assay to be used in high-throughput fashion using 96-well plates, making it a viable option for drug discovery efforts, particularly when the drugs in question have some level of mitotoxicity. Importantly, results obtained by Hoechst/PI staining assay show increased consistency, both with trypan blue exclusion results and between biological replicates when the assay is compared to other methods.

The protocol describes a rapid, high-throughput, reliable, inexpensive, and unbiased assay for efficiently determining cellular viability. This assay is particularly useful when cells&#39; mitochondria have been damaged, which interferes with other assays. The assay uses automated counting of cells stained with two nuclear dyes &#8211; Hoechst 33342 and propidium iodide.

The contribution of mitochondria to oncogenic transformation is a subject of wide interest and active study. As the field of cancer metabolism becomes ...

Cancer Research

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.

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 ...

Use of a Caspase Multiplexing Assay to Determine Apoptosis in a Hypothalamic Cell Model

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and decreases waste in costs and reagents. Here we describe optimization of a multiplex assay to assess apoptosis following a palmitic acid (PA) challenge in an in vitro hypothalamic model, using both fluorescent and luminescent based assays to measure viable cell counts and caspase-3/7 activity in a 96-well microtiter plate format. Following PA challenge, viable cells were determined by a resazurin-based fluorescent assay. Caspase-3/7 activity was then determined using a luminogenic substrate, DEVD, and normalized to cell number. This multiplexing assay is a useful technique for determining change in caspase activity following an apoptotic stimulus, such as saturated fatty acid challenge. The saturated fatty acid PA can increase hypothalamic oxidative stress and apoptosis, indicating the potential importance of assays such as that described here in studying the relationship between saturated fatty acids and neuronal function.

Multiplex assays can provide beneficial information for basic cellular mechanisms&#160;and eliminate waste of reagents and unnecessary repetitive experiments. We describe here a multiplex caspase-3/7 activity assay, using fluorescent- and luminescent-based methods, to determine cell viability in an in vitro hypothalamic model following oxidative challenge with palmitic acid.

The ability to multiplex assays in studies of complex cellular mechanisms eliminates the need for repetitive experiments, provides internal controls, and ...

Assessment of Mitochondrial Functions and Cell Viability in Renal Cells Overexpressing Protein Kinase C Isozymes

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-&epsilon; improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-&alpha; and PKC-&epsilon; are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-&epsilon; mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC.
Here, we present methods for assessing the role of PKC-&epsilon; in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-&epsilon; is activated by overexpressing the constitutively active PKC-&epsilon; mutant. PKC-&epsilon; is inhibited by overexpressing the inactive mutant of PKC-&epsilon;. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis.
These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

The effects of activation of protein kinase C (PKC) isozymes on mitochondrial functions associated with respiration and oxidative phosphorylation and on cell viability are described. The approach adapts adenoviral technique to selectively overexpress PKC isozymes in primary cell culture and a variety of assays to determine mitochondrial functions and energy status of the cell.

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC ...

Viability Assays for Cells in Culture

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16&#160;bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (&#945;-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.

Therapeutic compounds are often first examined in vitro with viability assays. Blind cell counts by a human observer can be highly sensitive to small changes in cell number but do not assess function. Computerized viability assays, as described here, can assess both structure and function in an objective manner.

Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized ...

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

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important checkpoints of apoptosis, including mitochondrial fragmentation, release of mitochondrial cytochrome c into the cytosol, activation of caspases, chromatin condensation, DNA damage, nuclear fragmentation, plasma membrane blebbing, cell shrinkage, cell surface exposure of phosphatidylserine, and formation of apoptotic bodies. Anastasis can occur when apoptotic stimuli are removed prior to death, thereby allowing dying cells to reverse apoptosis and potentially other death mechanisms. Therefore, anastasis appears to involve physiological healing processes that could also sustain damaged cells inappropriately. The functions and mechanisms of anastasis are still unclear, hampered in part by the limited tools for detecting past events after the recovery of apparently healthy cells. Strategies to detect anastasis will enable studies of the physiological mechanisms, the hazards of undead cells in disease pathology, and potential therapeutics to modulate anastasis. Here, we describe effective strategies using live cell microscopy and a mammalian caspase biosensor&#160;for identifying and tracking anastasis in mammalian cells.

The term anastasis refers to the phenomenon in which dying cells reverse a cell suicide process at a late stage, repair themselves, and ultimately survive. Here we demonstrate protocols for detecting and tracking cells that undergo anastasis.

Anastasis (Greek for &#8220;rising to life&#8221;) refers to the recovery of dying cells. Before these cells recover, they have passed through important ...

An Introduction to Cell Death

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as cell injury, low nutrient levels, or signaling proteins. Whereas necrosis is considered to be an &#8220;accidental&#8221; or unexpected form of cell death, evidence exists that apoptosis and autophagy are both programmed and &#8220;planned&#8221; by cells.In this introductory video, JoVE highlights key discoveries pertaining to cell death, including recent work done in worms that helped identify genes involved in apoptosis. We then explore questions asked by scientists studying cell death, some of which look at different death pathways and their interactions. Finally, several methods to assess cell death are discussed, and we note how researchers are applying these techniques in their experiments today.

Cell Death: Apoptosis, Autophagy and Necrosis

Necrosis, apoptosis, and autophagic cell death are all manners in which cells can die, and these mechanisms can be induced by different stimuli, such as ...

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Cell Biology

Annexin V and Propidium Iodide Labeling

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death&#8212;either necrosis or apoptosis. This technique relies on two components. The first, annexin V, is a protein that binds certain phospholipids called phosphatidylserines, which normally occur only in the inner, cytoplasm-facing leaflet of a cell&#8217;s membrane, but become &#8220;flipped&#8221; to the outer leaflet during the early stages of apoptosis. The second component is the DNA-binding dye molecule PI, which can only enter cells when their membranes are ruptured&#8212;a characteristic of both necrosis and late apoptosis.This video article begins with a review of the concepts behind annexin V and PI staining, and emphasizes how differential patterns of staining can be used to distinguish between cells progressing down different death pathways. We then review a generalized protocol for this technique, followed by a description of how researchers are currently using annexin V and PI staining to better understand cell death.

Cell Death Pathways and Annexin V &amp; PI Labeling studies

Staining with annexin V and propidium iodide (PI) provides researchers with a way to identify different types of cell death&#8212;either necrosis or ...

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

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