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 &amp;#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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27.9K 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 &#946;-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 ...

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 ...

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 Step-By-Step Method to Detect Neutralizing Antibodies Against AAV using a Colorimetric Cell-Based Assay

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. 
In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to &gt;10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in &gt;1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

A comprehensive laboratory protocol and analysis workflow are described for a rapid, cost-effective, and straightforward colorimetric cell-based assay to detect neutralizing elements against AAV6.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health ...

Histology Basics and Cell Death Detection in Honeybee Tissue

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather changes, various pesticides, pathogens, and malnutrition, mainly entering through the mouth and primarily affecting the digestive tracts of adult bees. To understand and prevent the effects of such external and internal stressors on honeybees, one useful research method is the immunohistochemical method. A basic protocol is described to prepare the midgut (ventriculus) and hypopharyngeal glands (HPGs) of adult bees for histological analysis. A detailed methodology is described to assess the level of cell damage and distinguish necrosis from programmed cell death (apoptosis) as a natural process of tissue regeneration. The results of adult honeybee treatment with oxalic acid and pesticides (insecticide and acaricide) and the determination of cell death in the ventriculus and HPGs are presented. The pros and cons of the methodology are also discussed.

Immunohistochemical methods are useful in honeybee research to detect and assess the level of apoptosis and necrosis in the midgut and hypopharyngeal glands of adult bees.

Honeybees (Apis mellifera L.) inside the hive (nurse workers and other hive bees) and outside the hive (foragers) are exposed to climate and weather ...

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous research indicated that multiple pathways could mediate the spontaneous death of neutrophils. A cocktail was developed by simultaneously targeting these pathways, caspases-lysosomal membrane permeabilization-oxidant-necroptosis inhibition plus granulocyte colony-stimulating factor (CLON-G), which prolonged the neutrophil lifespan to greater than 5 days without significantly compromising the neutrophil function. Concurrently, a reliable and stable protocol for assessing and evaluating neutrophil death was also developed. In this work, we show that CLON-G can prolong the neutrophil lifespan in vitro to more than 5 days, and we exhibit the lengthening of the neutrophil lifespan with FACS and confocal fluorescence microscopy. This report introduces procedures for the preparation of CLON-G and showcases an in vitro spontaneous death assay of neutrophils, which can be used for the study of neutrophils and for subsequently interrogating neutrophil death, thus providing a reliable resource for the neutrophil community.

Neutrophil Lifespan Extension with CLON-G and an In Vitro Spontaneous Death Assay

This protocol details the preparation of CLON-G to extend the neutrophil lifespan to greater than 5 days and provides a reliable procedure for evaluating neutrophil death with flow cytometry and confocal fluorescence microscopy.

The average lifespan of a neutrophil is less than 24 h, which limits basic research on neutrophils and the application of neutrophil studies. Our previous ...

Quantifying Cell Death Initiation Through Continuous Imaging

Studying Cell Death Initiation Using a Digital Microscope

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as the formation of cell death zones on inoculated leaves. Here, a protocol for studying the rate of cell death initiation by imaging inoculated leaves in the time between the cell death initiation and the cell death appearance using a digital microscope is presented. The digital microscope enables a continuous imaging process in desired intervals, which allows an accurate determination of cell death initiation rate up to minutes exactly, as opposed to hours in traditional methods. Imaging with the digital microscope is also independent of light and can therefore be used during day and night without disturbing the circadian rhythm of the plant. Different pathosystems resulting in programmed cell death development could be studied using this protocol with minor modifications. Overall, the protocol thus allows simple, accurate, and inexpensive identification of cell death initiation rate.

Author Spotlight: A Streamlined Approach to Studying Cell Death Initiation in Hypersensitive Response 

Here we present a protocol for studying the rate of programmed cell death initiation by continuously imaging inoculated leaves following cell death induction.

Hypersensitive response (HR)-conferred resistance is an effective defense response that can be determined by the N resistance genes. HR is manifested as ...