Name: pattern-triggered oxidative burst and seedling growth inhibition assays in arabidopsis thaliana
Uploaded: 2019-05-21T13:30:00
Description: Watch this Scientific Journal Video about Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana at JoVE.com

Work in our lab is funded through the Natural Resources and Engineering Research Council of Canada (NSERC) Discovery Program, the Canadian Foundation for Innovation John R. Evans Leader&#39;s Fund, and Queen&#39;s University. KS and IS are supported by tandem Ontario Graduate Scholarships and NSERC Canada Graduate Scholarships for master&#39;s students (CGS-M).

1. Detection of ROS burst in Arabidopsis leaf discs following immune elicitation
<ol>
	<li>Plant growth and maintenance.
	<ol>
		<li>To synchronize germination, stratify Arabidopsis seeds by suspending approximately 50 seeds in 1 mL of sterile 0.1% agar [w/v] and store at 4 &#176;C (no light) for 3-4 days. 
		NOTE:&#160;Stratify a wild type background control (for example, Col-0) and genotypes with high and low immune outputs (for example,&#160;cpk28-1&#160;and&#160;bak1-5,&#160;</em...

This paper describes two methods for assaying pattern-triggered immune responses in Arabidopsis, offering quantitative approaches to evaluating immune output without the use of specialized equipment. In combination, pattern-triggered ROS and SGI can be used to assess early and late responses to microbe perception, respectively.
The major limitation of the oxidative burst assay is variability. For reasons that are not completely understood, absolute RLUs often differ by an order of mag...

To perceive and defend against pathogens, plants have evolved membrane-bound pattern recognition receptors (PRRs) that detect conserved microbial molecules outside the cell known as microbe-associated molecular patterns (MAMPs)1. The binding of MAMPs to their cognate PRRs initiates protein kinase-mediated immune signaling resulting in broad-spectrum disease resistance2. One of the earliest responses following PRR activation is the phosphorylation and activation of integral plasma membrane RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) proteins that catalyze the production of extracellular reactive oxygen species (ROS)3,4. ROS play an important role in establishing disease resistance, acting both as secondary messengers to propagate immune signaling as well as direct antimicrobial agents5. The first observation of an immune-elicited oxidative burst was described using potato tubers of cv. Rishiri following Phytophthora infestans inoculation6. ROS production has been evaluated in several plant species using leaf discs7, cell suspension cultures8, and protoplasts6. Described here is a simple method for assaying pattern-triggered ROS production in leaf discs of Arabidopsis thaliana (Arabidopsis).
As a response to MAMP perception, activated RBOH proteins catalyze the production of superoxide radicals (O2-), hydroxyl radicals (&#8226;OH), and singlet oxygen (1O2) that are converted into hydrogen peroxide (H2O2) in the extracellular space9. H2O2 can be quantified by luminol-based chemiluminescence in the presence of the oxidizing agent horseradish peroxidase (HRP)10. HRP oxidizes H2O2 generating a hydroxide ion (OH&#8722;) and oxygen gas (O2) which react with luminol to produce an unstable intermediate that releases a photon of light10. Photon emission can be quantified as relative light units (RLUs) using a microplate reader or imager capable of detecting luminescence, which have&#160;become standard pieces of equipment in most molecular laboratories. By measuring the light produced over a 40-60-minute interval, a transient oxidative burst can be detected as early as 2-5 minutes after the elicitor treatment, peaking at 10-20 minutes, and returning to basal levels after ~60 minutes11. The cumulative light produced over this time course can be used as a measure of immune strength corresponding to the activation of RBOH proteins12. Conveniently, this assay does not require specialized equipment or cumbersome sample preparation.
Peaking shortly after MAMP detection, the oxidative burst is considered an early immune response, along with MAPK activation and ethylene production5. Later immune responses include transcriptional reprogramming, stomatal closure, and callose deposition2,5. Prolonged exposure to MAMPs continually activates energetically-costly immune signaling resulting in the inhibition of plant growth, indicative of a trade-off between development and immunity13. Pattern-triggered seedling growth inhibition (SGI) is widely used to assess immune output in Arabidopsis and has been integral to the identification of several key components of immune signaling including PRRs14,15,16. Therefore, this paper additionally presents an assay for&#160;pattern-triggered SGI in Arabidopsis, whereby seedlings are grown in multi-well plates containing standard media or media supplemented with an immune elicitor for 8-12 days and then weighed using an analytical scale.
To demonstrate how ROS and SGI assays can be used to monitor PRR-mediated signaling, three genotypes that represent varying immune outputs were chosen: (1) the wild type Arabidopsis accession Columbia (Col-0), (2) the dominant-negative bak1-5 mutant in which the multi-functional PRR co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1&#160;(BAK1) is non-functional in immune signaling17,18, and (3) the recessive cpk28-1 mutant, which lacks the regulatory protein CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) and displays heightened immune-triggered responses19,20. ROS and SGI assays are presented in response to a synthetically-produced elf18 peptide epitope of bacterial Elongation Factor Tu (EF-Tu), recognized in Arabidopsis by the PRR EF-Tu RECEPTOR (EFR)15. These protocols can be used with other immune elicitors such as the bacterial motility protein flagellin14 or endogenous Plant Elicitor Proteins (AtPeps)16, however, it should be noted that plant responsiveness differs depending on the elicitor21. Together, ROS and SGI assays can be used for the quick and quantitative assessment&#160;of early and late PRR-mediated responses.

<table>
	<tbody>
		<tr>
			<td>20-20-20 Fertilizer</td>
			<td>Plant Prod</td>
			<td>10529</td>
			<td>Mix 1g/L in water and apply to plants every 2 weeks for optimal growth.</td>
		</tr>
		<tr>
			<td>4 mm Biopsy Punch</td>
			<td>Medical Mart</td>
			<td>232-33-34-P</td>
			<td>A cork borer set with a 0.125 cm^2 surface area can also be used.</td>
		</tr>
		<tr>
			<td>48-Well Sterile Plates with Lid</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Scale with Draft Sheid</td>
			<td>VWR</td>
			<td>VWR-225AC</td>
			<td>Any standard analytical scale can be used for growth inibition assays, however, a direct computer output is optimal.</td>
		</tr>
		<tr>
			<td>BioHit mLine Mechanical 12 Multichannel Pipette (30-300 uL)</td>
			<td>Sartorius</td>
			<td>725240</td>
			<td>Any multichannel pipette can be used, as can a single pipetter if necessary.</td>
		</tr>
		<tr>
			<td>elf18 (Ac-SKEKFERTKPHVNVGTIG)</td>
			<td>EZ Biolab</td>
			<td>cp7211</td>
			<td>Store 10 mM stock peptide at -80C in low protein binding tubes. When thawed, store 100 uM working stock at -20C.</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>22-327379</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish Peroxidase</td>
			<td>Sigma-Aldrich</td>
			<td>P6782</td>
			<td>Dissolve in pure water. Store at -20C and away from light.</td>
		</tr>
		<tr>
			<td>Luminol</td>
			<td>Sigma-Aldrich</td>
			<td>A8511</td>
			<td>Dissolve in DMSO. Store at -20C and away from light.</td>
		</tr>
		<tr>
			<td>Murisage and Skoog Basal Salts</td>
			<td>Cedarlane Labs</td>
			<td>MSP09-100LT</td>
			<td>Store at 4C.</td>
		</tr>
		<tr>
			<td>Soil</td>
			<td>SunGrow Horticulture</td>
			<td>Sunshine Mix #1</td>
			<td>Other soil types can also be used to grow Arabidopsis. Mix with water when filling pots.</td>
		</tr>
		<tr>
			<td>SpectraMax Paradigm Multi Mode Microplate Reader with LUM Module</td>
			<td>Molecular Devices</td>
			<td>Must request a quote</td>
			<td>Any plate reader capable of detecting luminescence can be used for these assays.</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S0389-1KG</td>
			<td>Store at room temperature.</td>
		</tr>
		<tr>
			<td>White Polystyrene 96-Well Plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-589</td>
			<td></td>
		</tr>
	</tbody>
</table>

pattern-triggered oxidative burst and seedling growth inhibition assays in arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure the strength of immune activation in Arabidopsis thaliana following treatment with elicitor molecules. Presented first is a method for capturing the rapidly-induced and dynamic oxidative burst, which can be monitored using a luminol-based assay. Presented second is a method describing how to measure immune-induced inhibition of seedling growth. These protocols are fast and reliable, do not require specialized training or equipment, and are widely used to understand the genetic basis of plant immunity.

Mutant cpk28-119,25 and bak1-517,18 plants were used to demonstrate expected outcomes for genotypes with high and low immune responses, respectively, in oxidative burst and SGI assays relative to a wild-type background control (Col-0). To assess dose-dependent effects, a 10-fold peptide dilution series (1-1,000 nM) of elf18 was used. As expected, cpk2...

Watch this Scientific Journal Video about Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana at JoVE.com

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

This paper describes two methods for quantifying defense responses in Arabidopsis thaliana following exposure to immune elicitors: the transient oxidative burst, and the inhibition of seedling growth.

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...

These assays can be used to assess and compare immune outputs of different plant genotypes. The main advantage of these techniques is that they are quick, reliable, and use common laboratory equipment. For the growth inhibition assay, seedlings of the same size and age should be transplanted and fully blotted dry before weighing to limit variation.At four to five weeks post-germination, use a sharp four-millimeter biopsy punch to remove one leaf disc per Arabidopsis plant, avoiding the midvein and being careful to limit wounding. Collect the discs into individual wells of an unused 96-well luminometer plate containing 100 microliters of double-distilled water per well, adaxial side up, to prevent desiccation. If assessing multiple elicitors, remove a second leaf disc from the same leaf for each elicitor treatment, and cover the plate with the lid to allow the leaf discs to recover overnight at room temperature.The next morning, set the microplate reader parameters to a 1, 000-millisecond integration time in two-minute intervals over a 40-to 60-minute period to capture the dynamic oxidative burst. Next, use a multi-channel pipette to replace the water in each well with 100 microliters of freshly prepared reaction solution, including a control, no elicitor reaction for each genotype to assess the basal reaction oxygen species levels in the absence of elicitation. Then, immediately measure the light emission for all of the wavelengths in the visible spectrum on the microplate reader.At four days post-germination, load each well of a 48-well plate with 500 microliters of MS medium supplemented with a dilution of elicitor peptide. Next, use sterile forceps to carefully transplant six to eight seedlings of the same size, age, and genotype to each peptide dilution, taking care that there is no damage to the seedling or breakage to the root and that the root is submerged in medium. When all of the seedlings have been plated, seal the plates with Micropore tape, and place the plants under standard short-day conditions for eight to 12 days.To determine the percent growth inhibition, take a photo to visually record the growth inhibition before carefully removing the seedlings from each well. Then, dab the roots on a paper towel to dry, and weigh each seedling on an analytical scale. In this representative experiment, mutant plants with hyperactive immune signaling demonstrated a higher cumulative and average oxidative burst compared to wild-type Arabidopsis plants, whereas mutant plants with impaired immune signaling displayed a reduced oxidative burst at concentrations between 10 and 1, 000 nanomolar.Expected differences in seedling growth inhibition could also be discerned between all of the genotypes grown in 100 and 1, 000 nanomolar elicitor peptide concentrations. When grown in the 1, 000 nanomolar peptide dilution, the mutants with hyperactive immune signaling were markedly smaller than wild-type plants grown under the same conditions, while the mutants with impaired immune signaling displayed a weak inhibition of growth relative to wild-type plants. These methods provide evidence for early and late immune responses.Several other techniques, including MAP kinase assays, pathogen-induced gene expression, and infection assays, can be used to delineate these pathways. These techniques have been modified for use in large-scale screens, which have successfully identified pathogen receptor proteins and several other signaling components.

pattern-triggered-oxidative-burst-seedling-growth-inhibition-assays

Research

JoVE Journal

Immunology and Infection

Quantification of the Respiratory Burst Response as an Indicator of Innate Immune Health in Zebrafish

The phagocyte respiratory burst is part of the innate immune response to pathogen infection and involves the production of reactive oxygen species (ROS). ROS are toxic and function to kill phagocytized microorganisms. In vivo quantification of phagocyte-derived ROS provides information regarding an organism's ability to mount a robust innate immune response. Here we describe a protocol to quantify and compare ROS in whole zebrafish embryos upon chemical induction of the phagocyte respiratory burst. This method makes use of a non-fluorescent compound that becomes fluorescent upon oxidation by ROS. Individual zebrafish embryos are pipetted into the wells of a microplate and incubated in this fluorogenic substrate with or without a chemical inducer of the respiratory burst. Fluorescence in each well is quantified at desired time points using a microplate reader. Fluorescence readings are adjusted to eliminate background fluorescence and then compared using an unpaired t-test. This method allows for comparison of the respiratory burst potential of zebrafish embryos at different developmental stages and in response to experimental manipulations such as protein knockdown, overexpression, or treatment with pharmacological agents. This method can also be used to monitor the respiratory burst response in whole dissected kidneys or cell preparations from kidneys of adult zebrafish and some other fish species. We believe that the relative simplicity and adaptability of this protocol will complement existing protocols and will be of interest to researchers who seek to better understand the innate immune response.

The innate immune response protects organisms against pathogen infection. A critical component of the innate immune response, the phagocyte respiratory burst, generates reactive oxygen species that kill invading microorganisms. We describe a respiratory burst assay that quantifies reactive oxygen species produced when the innate immune response is chemically induced.

The phagocyte respiratory burst is part of the innate immune response to  pathogen infection and involves the production of reactive oxygen species (ROS). ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

Bioenergetics and the Oxidative Burst: Protocols for the Isolation and Evaluation of Human Leukocytes and Platelets

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and neurodegenerative diseases but assessing changes in bioenergetic function in patients is challenging.&#160;Although diseases such as diabetes or atherosclerosis present clinically with specific organ impairment, the systemic components of the pathology, such as hyperglycemia or inflammation, can alter bioenergetic function in circulating leukocytes or platelets. This concept has been recognized for some time but its widespread application has been constrained by the large number of primary cells needed for bioenergetic analysis.&#160;This technical limitation has been overcome by combining the specificity of the magnetic bead isolation techniques, cell adhesion techniques, which allow cells to be attached without activation to microplates, and the sensitivity of new technologies designed for high throughput microplate respirometry.&#160;An example of this equipment is the extracellular flux analyzer. Such instrumentation typically uses oxygen and pH sensitive probes to measure rates of change in these parameters in adherent cells, which can then be related to metabolism. Here we detail the methods for the isolation and plating of monocytes, lymphocytes, neutrophils and platelets, without activation, from human blood and the analysis of mitochondrial bioenergetic function in these cells. In addition, we demonstrate how the oxidative burst in monocytes and neutrophils can also be measured in the same samples. Since these methods use only 8-20 ml human blood they have potential for monitoring reactive oxygen species generation and bioenergetics in a clinical setting.

Blood leukocytes and platelets can be used as a marker of overall bioenergetic health of an individual and so have the potential to monitor pathological processes and the impact of treatments. Here&#160;we describe a method to isolate and measure mitochondrial function and the oxidative burst in these cells.

Mitochondrial dysfunction is known to play a significant role in a number of pathological conditions such as atherosclerosis, diabetes, septic shock, and ...

Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H2O2 into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H2O2 compared to a no H2O2 control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H2O2. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism ...

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Measuring Granulocyte and Monocyte Phagocytosis and Oxidative Burst Activity in Human Blood

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides the necessary methodology to add this assay to an exercise immunology arsenal. The first step in this assay is to prepare two aliquots ("H" and "F") of whole blood (heparin). Then, dihydroethidium is added to the H aliquot, and both aliquots are incubated in a warm water bath followed by a cold water bath. Next, Staphylococcus aureus (S. aureus) is added to the H aliquot and fluorescein isothiocyanate-labeled S. aureus is added to the F aliquot (bacteria:phagocyte = 8:1), and both aliquots are incubated in a warm water bath followed by a cold water bath. Then, trypan blue is added to each aliquot to quench extracellular fluorescence, and the cells are washed with phosphate-buffered saline. Next, the red blood cells are lysed, and the white blood cells are fixed. Finally, a flow cytometer and appropriate analysis software are used to measure granulocyte and monocyte phagocytosis and OB activity. This assay has been used for over 20 years. After heavy and prolonged exertion, athletes experience a significant but transient increase in phagocytosis and an extended decrease in OB activity. The post-exercise increase in phagocytosis is correlated with inflammation. In contrast to normal weight individuals, granulocyte and monocyte phagocytosis is chronically elevated in overweight and obese participants, and is modestly correlated with C-reactive protein. In summary, this flow cytometry-based assay measures the phagocytosis and OB activity of phagocytes and can be used as an additional measure of exercise- and obesity-induced inflammation.

The purpose of this manuscript is to present a method for measuring monocyte and granulocyte phagocytosis and oxidative burst activity in human blood samples.

The granulocyte and monocyte phagocytosis and oxidative burst (OB) activity assay can be used to study the innate immune system. This manuscript provides ...

Screening Assays to Characterize Novel Endothelial Regulators Involved in the Inflammatory Response

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory response by the endothelial layer is crucial to efficiently fight against harmful inputs and aid in the recovery of damaged areas. When the endothelial cells are exposed to an inflammatory environment, such as the outer component of gram-negative bacteria membrane, lipopolysaccharide (LPS), they express soluble pro-inflammatory cytokines, such as Ccl5, Cxcl1 and Cxcl10, and trigger the activation of circulating leukocytes. In addition, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 on the endothelial surface enables the interaction and adhesion of the activated leukocytes to the endothelial layer, and eventually the extravasation towards the inflamed tissue. In this scenario, the endothelial function must be tightly regulated because excessive or defective activation in the leukocyte recruitment could lead to inflammatory-related disorders. Since many of these disorders do not have an effective treatment, novel strategies with a focus on the vascular layer must be investigated. We propose comprehensive assays that are useful to the search of novel endothelial regulators that modify leukocyte function. We analyze endothelial activation by using specific expression targets involved in leukocyte recruitment (such as, cytokines, chemokines, and adhesion molecules) with several techniques, including: real-time quantitative polymerase chain reaction (RT-qPCR), western-blot, flow cytometry and adhesion assays. These approaches determine endothelial function in the inflammatory context and are very useful to perform screening assays to characterize novel endothelial inflammatory regulators that are potentially valuable for designing new therapeutic strategies.

Vascular endothelium tightly controls leukocyte recruitment. Inadequate leukocyte extravasation contributes to human inflammatory diseases. Therefore, searching for novel regulatory elements of endothelial activation is necessary to design improved therapies for inflammatory disorders. Here, we describe a comprehensive methodology to characterize novel endothelial regulators that can modify leukocyte trafficking during inflammation.

The endothelial layer is essential for maintaining homeostasis in the body by controlling many different functions. Regulation of the inflammatory ...

Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known as quasispecies, owing to their high mutation rate. Here, we describe how to measure the specific growth rate and the cell-binding ability of rotavirus as its phenotypes. Rotavirus is treated with trypsin to recognize the cell receptor and then inoculated into MA104 cell culture. The supernatant, including viral progenies, is collected intermittently. The plaque assay is used to confirm the virus titer (plaque-forming unit: pfu) of each collected supernatant. The specific growth rate is estimated by fitting time-course data of pfu/mL to the modified Gompertz model. In the assay of cell-binding, MA104 cells in a 24-well plate are infected with rotavirus and incubated for 90 min at 4 &#176;C for rotavirus adsorption to cell receptors. A low temperature restrains rotavirus from invading the host cell. After washing to remove unbound virions, RNA is extracted from virions attached to cell receptors followed by cDNA synthesis and reverse-transcription quantitative PCR (RT-qPCR). These protocols can be applied for investigating the phenotypic differences among viral strains.

Here we present two protocols, one for measuring the specific growth rate and the other for the cell-binding ability of rotavirus using the plaque assay and RT-qPCR. These protocols are available for confirming the differences in phenotypes between rotavirus strains.

Rotavirus is the main etiological factor for infantile diarrhea. It is a double-stranded (ds) RNA virus and forms a genetically diverse population, known ...

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, rhizosphere colonization by plant growth-promoting bacteria (PGPB) can increase the health or the development of host plants relative to uncolonized plants. However, in field settings, bacterial treatments with PGPB often do not provide substantial benefits to crops. One explanation is that this may be due to loss of the PGPB during interactions with endogenous soil microbes over the lifespan of the plant. This possibility has been difficult to confirm, since most studies focus on the initial colonization rather than maintenance of PGPB within rhizosphere communities. It is hypothesized here that the assembly, coexistence, and maintenance of bacterial communities are shaped by deterministic features of the rhizosphere microenvironment, and that these interactions may impact PGPB survival in native settings. To study these behaviors, a hydroponic plant-growth assay is optimized using Arabidopsis thaliana to quantify and visualize the spatial distribution of bacteria during initial colonization of plant roots and after transfer to different growth environments. This system&#8217;s reproducibility and utility are then validated with the well-studied PGPB Pseudomonas simiae. To investigate how the presence of multiple bacterial species may affect colonization and maintenance dynamics on the plant root, a model community from three bacterial strains (an Arthrobacter, Curtobacterium, and Microbacterium species) originally isolated from the A. thaliana rhizosphere is constructed. It is shown that the presence of these diverse bacterial species can be measured using this hydroponic plant-maintanence assay, which provides an alternative to sequencing-based bacterial community studies. Future studies using this system may improve the understanding of bacterial behavior in multispecies plant microbiomes over time and in changing environmental conditions.

Monitoring Bacterial Colonization and Maintenance on Arabidopsis thaliana Roots in a Floating Hydroponic System

Here we describe a hydroponic plant growth assay to quantify species presence and visualize the spatial distribution of bacteria during initial colonization of plant roots and after their transfer into different growth environments.

Bacteria form complex rhizosphere microbiomes shaped by interacting microbes, larger organisms, and the abiotic environment. Under laboratory conditions, ...

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are limited. In addition, they exhibit adverse side effects in certain groups of people. Therefore, discovery of novel therapeutics for clinical toxoplasmosis is imperative. The first step of novel antibiotic development is to identify chemical compounds showing high efficacy in inhibition of parasite growth using a high throughput screening strategy. As an obligate intracellular pathogen, Toxoplasma can only replicate within host cells, which prohibits the use of optical absorbance measurements as a quick indicator of growth. Presented here is a detailed protocol for a luciferase-based growth assay. As an example, this method is used to calculate the doubling time of wild-type Toxoplasma parasites and measure the efficacy of morpholinurea-leucyl-homophenyl-vinyl sulfone phenyl (LHVS, a cysteine protease-targeting compound) regarding inhibition of parasite intracellular growth. Also described, is a CRISPR-Cas9-based gene deletion protocol in Toxoplasma using 50 bp homologous regions for homology-dependent recombination (HDR). By quantifying the inhibition efficacies of LHVS in wild-type and TgCPL (Toxoplasma cathepsin L-like protease)-deficient parasites, it is shown that LHVS inhibits wild-type parasite growth more efficiently than &#916;cpl growth, suggesting that TgCPL is a target that LHVS binds to in Toxoplasma. The high sensitivity and easy operation of this luciferase-based growth assay make it suitable for monitoring Toxoplasma proliferation and evaluating drug efficacy in a high throughput manner.

Determination of Chemical Inhibitor Efficiency against Intracellular Toxoplasma Gondii Growth Using a Luciferase-Based Growth Assay

Presented here is a protocol to evaluate the inhibition efficacy of chemical compounds against in vitro intracellular growth of Toxoplasma gondii using a luciferase-based growth assay. The technique is used to confirm inhibition specificity by genetic deletion of the corresponding target gene. The inhibition of LHVS against TgCPL protease is evaluated as an example.

Toxoplasma gondii is a protozoan pathogen that widely affects the human population. The current antibiotics used for treating clinical toxoplasmosis are ...