Nicotiana benthamiana plants are grown in a temperature-controlled growth chamber at 25&deg;C . Fully expanded healthy leaves of 3-6 week old plants are used.
Tip: Better results are obtained by using newly emerging leaves
1. Agrobacterium transient infiltration protocol:
Day 1
<ol>
 <li>Streak LB/Rifampicin (25 &mu;g/ml)/Kanamycin (100 &mu;g/ml) agar plates with glycerol stocks of Agrobacterium tumefaciens (strain LBA4404) containing the appropriate vectors with the gene (s) to be assayed for cell death and the vector containing the GUS cassette under a constitutive promoter. Always include an empty vector control as a negative control.</li>
 <li>Incubate at 28&deg;C for 2 days.</li> 
</ol>
Day 3
<ol>
 <li>Inoculate 2 ml of LB containing Rifampicin (25 &mu;g/ml) and Kanamycin (100 &mu;g/ml) with a single colony from each LB/Rifampicin/Kanamycin plate previously streaked.</li>
 <li>Incubate each culture by shaking at 28&deg;C for 24 hours at 200 rpm until maximum growth density is reached.</li>
</ol>
Tip: Clumping is sometimes observed, in which case cultures need to be thoroughly resuspended (i.e. pipetting up and down) before proceeding.
Day 4
<ol>
<li>Following incubation, increase the total volume to 10 mL with fresh LB (containing the appropriate antibiotics) and acetosyringone (final concentration 25 &mu;M).</li>
<li>Incubate each culture by shaking at 28 &deg;C for another 16 hrs.</li>
</ol>
Day 5
<ol>
 <li>The following day wash each culture twice by adding 10 ml (do not add acetosyringone) infiltration medium (10 mM MgSO4.7H2O, 9 mM MES, pH 5.6) and centrifuge at 4000 x g for 10 min at room temperature.</li>
<li>After the final centrifugation step, resuspend each culture in 5 ml infiltration medium containing acetosyringone (100 &mu;M).</li>
<li>Measure the OD600nm, measurements between 0.1 and 0.9 are acceptable. Tip: OD600nm of as low as 0.1 have been used without a problem, however, higher ODs may produce more consistent results.</li>
<li>Incubate the cultures for 3 more hrs to prepare the Agrobacterium cultures for infection.</li>
<li>Mix cultures containing the gene(s) to be analyzed and the negative control (Agrobacterium containing the empty vector) in a 1:1 ratio with the culture containing the GUS cassette.</li>
<li>Infiltrate the abaxial (under) side of newly emerging leaves using a 1 ml needle-less syringe (Fig. 1). 
Tip: Infiltrate both the negative control mixture (empty vector + GUS cassette) and the mixture containing the gene to be assayed (gene x + GUS cassette) on opposite leaves of the same plant. Use triplicate plants for each treatment.
</li>
<li>3 days post-infiltration excise infiltrated leaves and assay for GUS protein expression.</li>
</ol>
2. Histochemical GUS assay
Day 8
<ol>
<li>Vacuum infiltrate the X-gluc substrate medium into the excised leaves.</li>
<li>Incubate in darkness at room temperature overnight or until distinct blue staining appears.</li>
</ol>
Day 9
<ol>
 <li>Rinse in distillated water.</li>
<li>Incubate in 70% ethanol until chlorophyll is removed, then transfer to distilled water again. Visually assess GUS expression levels. </li>
</ol>
3. Fluorometric MUG assay:
Day 8
<ol>
 <li>Isolate total protein by grinding infiltrated leaf discs in a mortar using liquid nitrogen. Add 100 uL of GUS extraction buffer (50 mM NaPi pH 7.0, 10 mM EDTA, 0.1% Triton X-100, 0.1 % N-lauroylsarcosine sodium salt, &beta;-mercaptoethanol (0.7 &mu;l/ml) while keeping the sample on ice.</li>
<li>Centrifuge suspension at 14,000 g for 5 min at 4 &deg;C and take supernatant as total soluble protein.</li> 
<li>Measure concentration by using a nanodrop according to manufacturer&#x2019;s instructions.</li>
<li>Adjust the protein concentration to 100 &mu;g of total soluble protein for each sample using GUS extraction buffer.</li>
<li>Add MUG substrate (4-methylumbelliferyl-&beta;-D-glucuronide trihydrate) prepared in GUS extraction buffer to a final concentration of 2 mM. A total volume of 100 &mu;L per sample (protein + MUG substrate) should be sufficient.</li>
<li>Incubate the reactions for 1 hr at 37 &deg;C.</li>
<li>Stop reactions by adding 800 &mu;l of GUS stop buffer (0.2 M Na2CO3).</li>
<li>Measure fluorescence using a plate reader at an excitation wavelength of 365 nm and emission wavelength of 455 nm and compare values to an MU standard curve. Report levels of GUS activity as nmol MU / &mu;g total soluble protein / min.</li> 
</ol>
4. Stocks and Solutions:
<ol>
<li>Acetosyringone (1 M) stock (FW = 196.2 g) &#x2013; 0.981 g in 5 mL of DMSO</li>
<li>Kanamycin (100 mg/ml) stock &#x2013; prepare in dH20 &#x2013; filter sterilize</li>
<li>Rifampicin (25 mg/ml) stock &#x2013; prepare in DMSO</li>
<li>Luria-Bertani (LB) liquid growth media: (1L) 
1 % (w/v) bacto-tryptone, 10 g 
0.5 % (w/v) bacto-yeast extract, 5 g 
170 mM sodium chloride 10 g 
pH - 7
</li>
<li>Acetosyringone (25 &mu;M) = 25 &mu;l of a 1 M stock in a final volume of 1 ml</li>
<li>Infiltration Medium: (1L) 
10 mM MgSO4.7dH20(FW = 246.48 g), 2.4648 g 
9 mM MES(FW = 213.25 g), 1.91925 g 
pH &#x2013; 5.6</li>
<li>Acetosyringone (100 &mu;M) = 100 &mu;l of a 1M stock in a final volume of 1 ml</li>
<li>GUS Extraction Buffer 
50 mM NaPi pH 7.0, 10 mM EDTA, 0.1 % Triton X-100, 0.1 % N-lauroylsarcosine sodium salt, &beta;-mercaptoethanol (0.7 &mu;l/ml)</li>
<li>2x Phosphate buffer 
0.2M NaH2PO4 and 0.2 M Na2HPO4 pH 7</li>
<li> X-Gluc substrate solution 
Dissolve 1 mg of 5-bromo-4-chloro-3-indolyl b-D-glucuronide (X-Gluc) in 0.1 ml methanol. Add 1 ml 2x phosphate buffer, 20 &mu;l 0.1M potassium ferricyanide, 10&mu;l Triton X-100 10% and 850 &mu;l of distillated water.</li>
</ol>
5. Representative results:
Following this protocol, loss of GUS expression is expected when cell death occurs. We have simultaneously expressed the reporter gene, &beta;-glucuronidase (GUS) with a member of the cyto-protective Arabidopsis Bcl-2 Associated athanoGene (BAG) family. We co-infiltrated N. benthamiana leaves with a 35S driven BAG expression cassette and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404. As shown in figure 2, a visible increase in GUS staining was observed following this infiltration. Conversely, when the known pro-apoptotic member of the Bcl-2 family BAX was used, a marked reduction of GUS staining was observed (Fig. 2). In both of these cases, GUS expression was visibly different compared to the control. However, when the difference in expression is less evident, fluorometric MUG assays can be performed to quanitate GUS expression.
<img src="/files/ftp_upload/2680/2680fig1.jpg" alt="Figure 1"/> 
Figure 1. Example of Agrobacterium mixture infiltration of the abaxial side of N. benthamiana leaves using a 1 ml needle-less syringe.
<img src="/files/ftp_upload/2680/2680fig2.jpg" alt="Figure 2"/> 
Figure 2. A GUS vector was co-expressed in N. benthamiana leaves with the anti-death gene, and a known inducer of cell death. GUS levels were compared to an empty vector control (GUS control).

<ol>
	<li>Jefferson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GUS+fusions:+,+&#946;-glucuronidase+as+a+sensitive+and+versatile+gene+fusion+marker+in+higher+plants.">GUS fusions: , &#946;-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.</a> EMBO J. 6, 3901-3901 (1987).</li><li>Nishihara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+&#946;-Glucuronidase+Gene+in+Pollen+of+Lily+(Lilium+longiflorum),+Tobacco+(Nicotiana+tabacum),+Nicotiana+rustica,+and+Peony+(Paeonia+lactiflora)+by+Particle+Bombardment.">Expression of the &#946;-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica, and Peony (Paeonia lactiflora) by Particle Bombardment.</a> Plant Physiol. 102, 357-357 (1993).</li><li>Hodal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 87, 115-115 (1992).</li><li>Kabbage, 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+BAG+proteins:+a+ubiquitous+family+of+chaperone+regulators.">The BAG proteins: a ubiquitous family of chaperone regulators.</a> Cell Mol. Life Sci. . 65, 1390-1390 (2008).</li><li>Kang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtBAG6,+a+novel+calmodulin-binding+protein,+induces+programmed+cell+death+in+yeast+and+plants.">AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants.</a> Cell Death Differ. 13 (1), 84-84 (2006).</li><li>Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 165 (1), 1-1 (2006).</li><li>Takayama, S., Reed, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+targeting+and+regulation+by+BAG+family+proteins.">Molecular chaperone targeting and regulation by BAG family proteins.</a> Nat Cell Biol. 3 (10), 237-237 (2001).</li><li>Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+functional+analysis+of+BAG-1:+a+novel+Bcl-2-binding+protein+with+anti-cell+death+activity.">Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity.</a> Cell. 80 (2), 279-279 (1995).</li><li>Vitha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+&#946;-glucuronidase+assay+in+transgenic+plants.">Quantitative &#946;-glucuronidase assay in transgenic plants.</a> Biol. Plant. 35, 151-151 (1993).</li><li>Otha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+Expression+in+Tobacco+of+a+&#946;-Glucuronidase+(GUS)+Reporter+Gene+Containing+an+Intron+Within+the+Coding+Sequence.">Construction and Expression in Tobacco of a &#946;-Glucuronidase (GUS) Reporter Gene Containing an Intron Within the Coding Sequence.</a> Plant Cell Physiol. 31, 805-805 (1990).</li></ol>

No conflicts of interest declared.

It is often difficult to use cell death detection techniques in plants that are common in mammalian systems. In combination with a GUS reporter system, we present a plant based, sensitive method for the detection and analysis of cell death players. This method takes advantage of the simple fact that live cells are required for GUS expression to occur. To ensure meaningful results and repeatability, it is critical that the cultures harboring the GUS cassette and the gene to be assayed are infiltrated at equal ratios. The...

<table border="1">
	<tbody>
 	<tr>	
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>	
 	<td>Rifampicin</td>
 <td>VWR</td>
 <td>IC19549001</td>
 <td>25mg/mL stock in DMSO</td>
 </tr>
 <tr>	
 	<td>4'-hydroxy-3',5'-dimethoxyacetophenone (Acetosyringone)</td>
 <td>VWR</td>
 <td>TCD2666</td>
 <td>0.981 g/mL in DMSO (1M stock)</td>
 </tr>
 <tr>	
 	<td>2-(4-morpholino)ethanesulfonic acid monohydrate (MES)</td>
 <td>VWR</td>
 <td>EM-6110</td>
 <td>1.92 g/L in water for making 1 L of infiltration media</td>
 </tr>
 <tr>	
 	<td>Bacto-tryptone</td>
 <td>Fisher</td>
 <td>BP1421-2</td>
 <td>10g/L in water for making 1 L of LB medium</td>
 </tr>
 <tr>	
 	<td>Bacto-yeast extract</td>
 <td>VWR</td>
 <td>EM1.03753.0500</td>
 <td>5g/L in water for making 1 L of LB medium</td>
 </tr>
 <tr>	
 	<td>Sodium chloride</td>
 <td>VWR</td>
 <td>EM-7710</td>
 <td>10g/L in water for making 1 L of LB medium</td>
 </tr>
 <tr>	
 	<td>Sodium phosphate monobasic monohydrate</td>
 <td>VWR</td>
 <td>MK-7868-12</td>
 <td>2.5g/L in water for making 50mM of buffer NaPi</td>
 </tr>
 <tr>	
 	<td>Sodium phosphate dibasic heptahydrate</td>
 <td>VWR</td>
 <td>EMD-SX0715-1</td>
 <td>5g/L in water for making 50mM of buffer NaPi</td>
 </tr>
 <tr>	
 	<td>Magnesium sulfate heptahydrate</td>
 <td>VWR</td>
 <td>EM-MX0070-1</td>
 <td>2.5 g/L in water for making 1 L of infiltration media</td>
 </tr>
 <tr>	
 	<td>N-Lauroylsarcosine</td>
 <td>VWR</td>
 <td>TCL0151-500G</td>
 <td>0.1% v/v in GUS buffer</td>
 </tr>
 <tr>	
 	<td>5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium salt (X-gluc)</td>
 <td>Gold Biotechnology</td>
 <td>G1281C</td>
 <td>1mg/100uL in methanol 100%</td>
 </tr>
 <tr>	
 	<td>4-Methylumbelliferyl-&mu;-D-glucuronide hydrate (MUG)</td>
 <td>Sigma</td>
 <td>M5664</td>
 <td>2 mM in 100 uL of GUS extraction buffer</td>
 </tr>
 <tr>	
 	<td>Potassium ferrocyanide trihydrate</td>
 <td>VWR</td>
 <td>EM-PX1460-1</td>
 <td>100mM stock for X-gluc substrate solution</td>
 </tr>
 <tr>	
 	<td>&beta;-Methylumbelliferone
(MU)
</td>
 <td>Sigma-Aldrich</td>
 <td>M1381</td>
 <td>For MU standard curve use GUS extraction buffer</td>
 </tr>
 <tr>	
 	<td>Sodium carbonate</td>
 <td>VWR</td>
 <td>EM-SX0395-11</td>
 <td>0.2 M in water for making GUS stop buffer</td>
 </tr>
 
 </tbody>
</table>

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.

Watch this Scientific Journal Video about A Î²-glucuronidase GUS Based Cell Death Assay at JoVE.com

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.

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 ...

a-glucuronidase-gus-based-cell-death-assay

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28.0K Views.  Texas A&M University. 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.

Video: A β-glucuronidase GUS Based Cell Death Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

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).

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 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 ...

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This technique has been widely exploited in tissue culture cells, and to a lesser extent, in primary tissue. The application of ChIP to rodent embryonic tissue, especially at early times of development, is complicated by the limited amount of tissue and the heterogeneity of cell and tissue types in the embryo. Here we present a method to perform ChIP using a dissociated embryonic day 8.5 (E8.5) embryo. Sheared chromatin from a single E8.5 embryo can be divided into up to five aliquots, which allows the investigator sufficient material for controls and for investigation of specific protein:chromatin interactions.
We have utilized this technique to begin to document protein:chromatin interactions during the specification of tissue-specific gene expression programs. The heterogeneity of cell types in an embryo necessarily restricts the application of this technique because the result is the detection of protein:chromatin interactions without distinguishing whether the interactions occur in all, a subset of, or a single cell type(s). However, examination of tissue-specific genes during or following the onset of tissue-specific gene expression is feasible for two reasons. First, immunoprecipitation of tissue specific factors necessarily isolates chromatin from the cell type where the factor is expressed. Second, immunoprecipitation of coactivators and histones containing post-translational modifications that are associated with gene activation should only be found at genes and gene regulatory sequences in the cell type where the gene is being or has been activated. The technique should be applicable to the study of most tissue-specific gene activation events.
In the example described below, we utilized E8.5 and E9.5 mouse embryos to examine factor binding at a skeletal muscle specific gene promoter. Somites, which are the precursor tissues from which the skeletal muscles of the trunk and limbs will form, are present at E8.5-9.54,5. Myogenin is a regulatory factor required for skeletal muscle differentiation 6-9. The data demonstrate that myogenin is associated with its own promoter in E8.5 and E9.5 embryos. Because myogenin is only expressed in somites at this stage of development 6,10, the data indicate that myogenin interactions with its own promoter have already occurred in skeletal muscle precursor cells in E8.5 embryos.

We demonstrate a chromatin immunoprecipitation (ChIP) method to identify factor interactions at tissue-specific genes during or after the onset of tissue-specific gene expression in mouse embryonic tissue. This protocol should be widely applicable for the study of tissue-specific gene activation as it occurs during normal embryonic development.

Chromatin immunoprecipitation (ChIP) is a powerful tool to identify protein:chromatin interactions that occur in the context of living cells 1-3. This ...

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 ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in the biogenesis of melanosomes, a lysosome-related organelle produced by melanocytes. REs deliver the melanocyte-specific cargo to premature melanosomes during their formation. Blockage in the generation of REs, observed in several mutants of Hermansky-Pudlak syndrome, results in hypopigmentation of skin, hair, and eye. Therefore, studying the dynamics (refer to number and length) of REs is useful to understand the function of these organelles in normal and disease conditions. In this study, we aim to measure the RE dynamics using a resident SNARE STX13.

Recycling endosomes are part of the endosomal tubular network. Here we present a method to quantify the dynamics of recycling endosomes using GFP-STX13 as an organelle marker.

Recycling endosomes (REs) are tubular-vesicular organelles generated from early/sorting endosomes in all cell types. These organelles play a key role in ...

A β-glucuronidase (GUS) Based Cell Death Assay

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Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

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

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

Chromatin Immunoprecipitation Assay for Tissue-specific Genes using Early-stage Mouse Embryos

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

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

The Microscopy-Based Assay to Study and Analyze the Recycling Endosomes using SNARE Trafficking