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 &mu;L 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 &mu;L siliconized tubes, reduce all volumes by 1/5.
The optimum concentration for flow cytometry analysis is 2-4 x 106 cells per 200 &mu;L 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 &mu;L 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 &mu;L 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 &mu;L of 1 x Annexin V binding buffer to each reaction tube. There should be approximately 200 &mu;L in each tube.</li>
	<li>Add 4 &mu;L of PI (Sigma, Cat# P-4864-10ML) that has been diluted 1:10 in 1 x Annexin V binding buffer (i.e. 1 &mu;L PI with 9 &mu;L 1 x Annexin V binding buffer). This will yield a final PI concentration of 2 &mu;g/mL in each sample.</li>
	<li>Incubate tubes in the dark for 15 minutes at room temperature.</li>
	<li>Add 500 &mu;L 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 &mu;L 1 x Annexin V binding buffer and 500 &mu;L 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&deg;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 &mu;L of 1:100 diluted RNase A (Sigma, R4642) to give a final concentration of 50 &mu;g/mL. Incubate for 15 min at 37&deg;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="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="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 &mu;g/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="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/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+labeled+Annexin+V.">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/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> 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Annexin+V+expression+in+apoptotic+peripheral+blood+lymphocytes:+An+electron+microscopic+evaluation.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flow+cytofluorometric+analysis+of+cell+cycle+distributions+using+propidium+iodide.+Properties+of+the+method+and+mathematical+analysis+of+the+data.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+DNA+of+Annexin+V-binding+apoptotic+cells+is+highly+fragmented.">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/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, 795-808 (1992).</li><li>Kroemer, G., Dallaporta, B., Resche-Rigon, 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+mitochondrial+death/life+regulator+in+apoptosis+and+necrosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+and+necrotic+cell+death+induced+by+death+domain+receptors.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caspases+disrupt+the+nuclear-cytoplasmic+barrier.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conventional+apoptosis+assays+using+propidium+iodide+generate+a+significant+number+of+false+positives+that+prevent+accurate+assessment+of+cell+death.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minireview:+Use+of+DNA-specific+anthraquinone+dyes+to+directly+reveal+cytoplasmic+and+nuclear+boundaries+in+live+and+fixed+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spectral+analysis+of+the+DNA+targeting+bisalkylaminoanthraquinone+DRAQ5+in+intact+living+cells.">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.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>1 x PBS-/-</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1 x Annexin V Binding Buffer</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>556454</td>
<td>&nbsp;</td>
<tr>
<td>Annexin V-Alexa Fluor 488</td>
<td>&nbsp;</td>
<td>Molecular Probes (Invitrogen)</td>
<td>A13201</td>
<td>&nbsp;</td>
<tr>
<td>Propidium iodide (PI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P4864</td>
<td>1 mg/mL in H2O</td>
</tr>
<tr>
<td>2% Formaldehyde</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>F1268</td>
<td>&nbsp;</td>
<tr>
<td>RNase A from bovine pancreas</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>R4642</td>
<td>&nbsp;</td>
<tr>
<td>DRAQ5</td>
<td>&nbsp;</td>
<td>Biostatus</td>
<td>DR50050</td>
<td>Dilute 1:60 in 1 x PBS-/-</td>
</tr>		</tbody>
		</table>
		</div>

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

102.8K Views.  University of Alberta. 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.

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

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

Interview: Bioreactors and Surfaced-Modified 3D-Scaffolds for Stem Cell Research

A Nature Editorial in 2003 asked the question "Good-bye, flat biology?" What does this question imply? In the past, many in vitro culture systems, mainly monolayer cultures, often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.
The last generation of CellChips -- micro-thermoformed containers -- a specific technology was developed, which offers the additional possibility to modify the whole surface of the 3D formed containers. This allows a surface-patterning on a submicron scale with distinct signalling molecules. Sensors and signal electrodes may be incorporated. Applications range from basic research in cell biology to toxicology and pharmacology. Using biodegradable polymers, clinical applications become a possibility. Furthermore, the last generation of micro-thermoformed chips has been optimized to allow for cheap mass production.

In the past many in vitro culture systems -- mainly monolayer cultures -- often suffered from the disadvantage that differentiated primary cells had a relatively short life-span and de-differentiated during culture. As a consequence, most of their organ-specific functions were lost rapidly. Thus, in order to reproduce better conditions for these cells in vitro, modifications and adaptations have been made to conventional monolayer cultures.

A Nature Editorial in 2003 asked the question  "Good-bye, flat biology?"  What does this question imply? In the past, many in vitro culture systems,  ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

A &#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs1,2,6,7. During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects3-5. To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells.
Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it8-10. After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death.
To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome11-13. The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible12,13. In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis14, is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase.
In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media12,13. Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.

We present a simple protocol to visualize regions of programmed cell death (PCD) in mouse embryos and differentiating embryonic stem (ES) cell cultures using a highly soluble dye called LysoTracker.

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9.
The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

Contribution of the ACF chromatin remodeling factor to E4orf4-induced cell death was measured. The protocol includes selection of cell clones in which doxycycline treatment induces conditional knockdown of the ACF subunits Acf1 and SNF2h, and use of the DAPI assay to measure E4orf4-induced cell death in the inducible cell lines.

Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

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 ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...