Name: a plate competition assay as a quick preliminary assessment of disease suppression
Uploaded: 2018-10-28T15:00:00.000+00:00
Duration: 5 min 23 s
Description: Watch this Scientific Journal Video about A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression at JoVE.com

The Vermont Agricultural Experiment Station Competitive Hatch Program VT-HO1609 funded the research. Lynn Fang used the method as part of her M.S. thesis at University of Vermont6.

1. Prepare in Advance
<ol>
	<li>Master fungal culture (test organism)
	<ol>
		<li>Order from the American Type Culture Collection, Microbiology Collection9, by its teleomorph Thanatephorus cucumeris (Frank) (ATCC 10154) or MYA 4031.</li>
		<li>Alternatively: Collect local isolates as done by the authors. Seedlings of red radish (Raphanus sativus) work well as a bait plant; collect soil from a location known to have a history of damping-off or root rot caused by R. solani. Sow seeds in the soil and isolate from root lesions when seedlings are 3 to 4 weeks old.
		<ol>
			<li>Remove seedlings from soil and rinse with tap water. Look for brown tissue in the hypocotyl region between the leaves and the root (Figure 1).</li>
			<li>Use a single edged razor blade to cut 1 cm segments of the hypocotyl or root that have a brown color. Use flame-sterilized forceps to dip the segments in 10% solution of household bleach for 1 min, followed by a 10 s rinse in sterile water. 
			NOTE: The top and bottom of a sterile Petri dish are useful containers for this step.</li>
			<li>Use flame-sterilized forceps to transfer the plant pieces to the inside of a paper towel to pat dry and then place on the surface of a Petri dish with water agar (15 g in 1 L).</li>
			<li>Place the Petri dish inside a plastic container with a lid and incubate at room temperature (approximately 20 &#176;C). Wipe the interior of the container with 10% bleach or 75% ethanol and let it air dry before inserting the dish.</li>
			<li>Keep the isolates in long-term storage on a minimal media of corn meal agar (17 g in 1 L) slants (at 5 &#176;C).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58767/58767fig1.jpg" /> 
Figure 1: Disease symptoms. Soil containing R. solani results in patchy germination and establishment (left). Symptoms on radish seedlings occur as brown lesions at the hypocotyl (right). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58767/58767fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="2">
	<li>Use potato dextrose agar plates (39 g in 1 L) for inoculum for this plate assay.
	<ol>
		<li>Establish a daughter culture of Rhizoctonia culture on potato dextrose agar one or two days before the assay, to insure it is well established on the culture plate before the assay. Start the daughter culture by transferring a small piece (10 mm diameter) of R. solani from the master culture to the center of a fresh plate of potato dextrose agar (Figure 2). 
		NOTE: Work in a laminar flow hood or wipe down a laboratory bench with 10% bleach or 75% ethanol to minimize risk of contamination.</li>
	</ol>
	</li>
	<li>Autoclave 25 mL test tubes with 10 mL aliquots of distilled water (2 x number of samples).</li>
</ol>
2. Preparation of Samples to Test
<ol>
	<li>Add two independent 0.5 g samples of each compost test sample to 10 mL of autoclaved water in 25 mL test tubes. Shake the test tubes&#160;overnight.
	<ol>
		<li>Label the test tubes with unique sample numbers, and each member of the pair as A (reference) or B (sample).</li>
	</ol>
	</li>
	<li>After 24 h, add 1.5 g of plain agar to 90 mL of distilled water in&#160;two 125 mLconical flasks.</li>
	<li>Autoclave both conical flasks with agar, and the A sample of each pair for 20 min with a slow exhaust setting.</li>
	<li>After the autoclave, place the conical flasks with agar into a 45 &#176;C water bath until they reach equilibrium (about 30 min). Do not put any sample slurries in the water bath until it has cooled to 45 &#176;C.</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58767/58767fig2.jpg" /> 
Figure 2: Illustration of protocol. Plugs of R. solani are placed on Petri dishes containing compost water extract. The diameter of mycelial growth is measured after 1-2 days with the aid of a stereo microscope to improve resolution and contrast. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58767/58767fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="5">
	<li>Pour the A sample (autoclaved reference) compost slurry into the molten agar of the A agar flask. Swirl to disperse compost into the agar.</li>
	<li>Pour the B sample (living sample) compost slurry into the molten agar of the B agar flask. Swirl to disperse compost into the agar.</li>
	<li>Pour the mixture of each flask into five plastic Petri dishes that are 100 mm in diameter.</li>
	<li>Let the plates cool overnight so the agar hardens.</li>
</ol>
3. Add the Rhizoctonia Challenge
<ol>
	<li>Using aseptic technique, transfer equal size pieces of R. solani to each pair of sample agar plates (3 to 5 mm corkborer works well to establish equal sized pieces). Take pieces of the colony from the outside margin of the colony to insure mycelium is actively growing. 
	NOTE: Work in a laminar flow hood or wipe down a laboratory bench with 10% bleach or 70% ethanol.</li>
	<li>Incubate the plates at room temperature for 1-2 days until the colony growth reaches about half way to the edge of the A plates.</li>
</ol>
4. Measure R. solani Growth
<ol>
	<li>Measure the radius of the mycelium in each plate to the nearest 1 mm with a clear flat ruler using a stereo microscope with transmitted illumination. 
	NOTE: Oblique or dark field illumination will make it easier to see and measure the transparent hyphae. At this point, zones of inhibition will be visible around compost fragments in the B plate if the compost is suppressive.</li>
	<li>Record the radius at three places per plate and compute a mean of the three to serve as a representative measure.</li>
	<li>Subtract the mean of the B plates from the A plates. If B &#60; A then there are microbes in the B plates that are suppressive to the R. solani pathogen.</li>
	<li>Divide the mean radius by the number of days of challenge to express units of relative suppression as a rate, mm mycelium per day.</li>
</ol>

<ol>
	<li>Gonzalez Garcia, G., Onco, M. A. P., Susan, V. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review.+Biology+and+systematics+of+the+form+genus+Rhizoctonia.">Review. Biology and systematics of the form genus Rhizoctonia.</a> Spanish Journal of Agricultural Research. 4 (1), 55-79 (2006).</li><li>Bonanomi, G., Antignani, V., Pane, C., Scala, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suppression+of+soilborne+fungal+diseases+with+organic+amendments.">Suppression of soilborne fungal diseases with organic amendments.</a> Journal of Plant Pathology. 89, 311-324 (2007).</li><li>Noble, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Risks+and+benefits+of+soil+amendment+with+composts+in+relation+to+plant+pathogens.">Risks and benefits of soil amendment with composts in relation to plant pathogens.</a> Australasian Plant Pathology. 40, 157-167 (2011).</li><li>Wichuk, K. M., McCartney, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compost+stability+and+maturity+evaluation+-+a+literature+review.">Compost stability and maturity evaluation - a literature review.</a> Canadian Journal of Civil Engineering. 37, 1505-1523 (2010).</li><li>Alfano, G., Lustrato, G., Lima, G., Vitullo, D., Ranalli, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+composted+olive+mill+wastes+to+predict+potential+plant+disease+suppressiveness.">Characterization of composted olive mill wastes to predict potential plant disease suppressiveness.</a> Biological Control. 3, 199-207 (2011).</li><li>Fang, 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> Biological indicators of compost-mediated disease suppression against the soilborne plant pathogen Rhizoctonia solani. , (2015).</li><li>Bonanomi, G., Antignani, V., Capodilupo, M., Scala, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+the+characteristics+of+organic+soil+amendments+that+suppress+soilborne+plant+diseases.">Identifying the characteristics of organic soil amendments that suppress soilborne plant diseases.</a> Soil Biology and Biochemistry. 42, 136-144 (2010).</li><li>Neher, D. A., Fang, L., Weicht, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecoenzymes+as+indicators+of+compost+to+suppress+Rhizoctonia+solani.">Ecoenzymes as indicators of compost to suppress Rhizoctonia solani.</a> Compost Science and Utilization. 25 (4), 251-261 (2017).</li><li>. ATCC&#174; Mycology Culture Guide Available from: <a target="_blank" href="https://www.atcc.org/~/media/PDFs/Culture%20Guides/Mycology_Guide.ashx">https://www.atcc.org/~/media/PDFs/Culture%20Guides/Mycology_Guide.ashx</a> (2018)</li><li>Berns, A. E., Phillip, H., Narres, H. -. D., Burauel, P., Vereecken, H., Tappe, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+gamma-sterilization+and+autoclaving+on+soil+organic+matter+structure+as+studied+by+solid+state+NMR,+UV+and+fluorescence+spectroscopy.">Effect of gamma-sterilization and autoclaving on soil organic matter structure as studied by solid state NMR, UV and fluorescence spectroscopy.</a> European Journal of Soil Science. 59, (2008).</li></ol>

The authors have nothing to disclose.

We know from previous research, that certain composts are effective at suppressing R. solani and that the suppressive effects are due to the microbes living in the compost, not the abiotic properties of compost6,8. The use of autoclaving as a means to &#39;kill&#39; microbiota has been criticized because it affects the carbon chemistry of the media10. We compared the use of autoclaving to vacuum filtration through Whatman No. 1 paper. Treatments that were neither filtered nor autoclaved showed the greatest suppression of R. solani growth6. Filtration removed the larger, solid particles that were apparently important to disease suppression by likely harboring antibiotic producing microbes.
Advantages of the method are the simplicity and affordability. Similar to the conclusions and recommendations of Alfano et al.2, the plate competition assay is a quick preliminary assessment of disease suppression but not reliable as a standalone assay because only the pathogen, and not the plant host, is present. Besides, disease would not establish if the environment was unfavorable; R. solani diseases are favored by warm and moist conditions1. Overall, there is more complexity in the soil and compost ecosystem than could be mimicked entirely by a single laboratory assay4,8. The plate assay is comparable to other indicators including a nematode index of compost maturity and three microbial ecoenzymes, phosphatase, &#946;-1,4-glucosidase and &#946;-1,4-N-acetylglucosaminidase8. The ecoenzymes reflects the state of decomposition and the carbon and nutrient needs of the microbial community. Confirmation of suppressiveness can be achieved with plant assays but they take longer to complete. Isolating and maintaining a pure culture of a fungus takes care to avoid contamination. ATCC9 offers tips and techniques for culturing yeasts and filamentous fungi.
The method is a potential candidate for commercial certification of disease suppressive properties. The result reflects suppression as a soil function. It does not specify mechanisms or specific microbial species responsible for disease suppression. A future application could be to cut out a portion of the inhibition zone for extraction of DNA for sequencing and identification of community members.

Rhizoctonia represents a broad complex of fungi, of which Thanatephorus cucumeris (Frank) Donk (anamorph = Rhizoctonia solani K&#252;hn) is the pathogen causing root rot and damping-off1. Rhizoctonia solani is an aggressive pathogen and a saprophyte that can survive as sclerotia under adverse environmental conditions1. As a result, it has a global distribution and can cause disease on wide range of plant hosts including Solanaceae, Fabaceae, Asteraceae, and Brassicaceae resulting in serious economic losses.
Compost has the capacity to harbor biocontrol agents for certain plant pathogens2. However, not all composts are alike nor do they affect all pathogens similarly3. Wood-based carbon has higher lignin to cellulose ratios than hay or straw-carbon based composts. R. solani prefers readily available carbon found in straw. In contrast, biological control fungi, such as Trichoderma spp., are more effective when carbon is less readily available. Beneficial fungi and bacteria into compost can suppress plant disease through competition, antagonism or regulating plant growth3. The proposed assay primarily detects antagonism created by production of antibiotics, ecoenzymes or chelators that are detrimental to the pathogen.
Plant bioassays are a gold standard to determine whether composts favor or deter plant growth4. However, plant bioassays are time-consuming (weeks to months) to complete which may be longer than desired and requires more labor to extract plants with roots to quantify severity of root systems. Comparably robust, but quicker (days) assays would be ideal for quality control programs. The goal of this paper is to demonstrate a relatively quick and accurate test to predict suppressive potential of compost. The method was patterned after Alfano et al.5 with two exceptions, compost extracts were diluted and water agar was used in place of potato dextrose agar (PDA). R. solani grows rapidly on simple water agar media whereas PDA promoted growth of bacteria and other fungi that contaminated the culture6.
This plate assay serves as an indicator of suppression that applies to a range of plant pathogens that survive in soil as saprophytes including R. solani7, Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot. The plate competition assay is useful to screen a range of compost products for containing communities of microbes that serve as biological control agents of soil pathogens. The assay was one of the most consistent indicators of disease suppression in commercial compost products6,8. Products were chosen for their variation in recipe, maturity, and production process.

<table><tbody><tr><td>autoclavable narrow-neck glass conical flask</td><td>Fisher Scientific</td><td>10-040D</td><td>125-mL</td></tr><tr><td>autoclavable glass testtubes</td><td>Fisher Scientific</td><td>14-925J</td><td>25-mL</td></tr><tr><td>dehydrated granulated agar</td><td>Fisher Scientific</td><td>DF0145-17-0</td><td>500 g quantity</td></tr><tr><td>heat resistant gloves</td><td>Fisher Scientific</td><td>MEMGG1314WL</td><td>several brands available</td></tr><tr><td>Parafilm (strips of 2-3 cm wide)</td><td>Fisher Scientific</td><td>PM992 13-374-16</td><td>5 or 10 cm widths work</td></tr><tr><td>disposable polystyrene petri dishes</td><td>Fisher Scientific</td><td>R80116</td><td>comes in sleeves of 20/ea or cases of 500</td></tr><tr><td>dehydrated potato dextrose agar</td><td>Fisher Scientific</td><td>DF0013-15-8</td><td>comes in quantities of 100, 500 and 2000 grams</td></tr><tr><td>benchtop reciprocal shaker</td><td>Thomas Scientific</td><td>1227Y31</td><td>other models will work</td></tr><tr><td>water bath</td><td>ThermoScientific</td><td>S37363</td><td>5L general purpose</td></tr><tr><td>clear ruler, flat, at least 10 cm</td><td>Any</td><td></td><td>use metric rule</td></tr><tr><td>ATCC culture</td><td>American Type Culture Collection</td><td>ATCC 10154</td><td>teleomorph &lt;em&gt;Thanatephorus cucumeris&lt;/em&gt; (Frank) (ATCC 10154) or MYA 4031;</td></tr><tr><td>lab tape</td><td>Fisher Scientific</td><td>15935</td><td>autoclavable and removable, 1&quot; wide preferred</td></tr><tr><td>water resistant marker</td><td>office or scientific supply</td><td>Sharpie fine tip</td><td>write sample number on tape</td></tr></tbody></table>

a plate competition assay as a quick preliminary assessment of disease suppression

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against soilborne fungus Rhizoctonia solani. R. solani is a pathogen of a wide range of plant hosts worldwide. The fungus survives in soils as a saprophyte and grows rapidly on simple water agar media. The plate assay is a rapid method to compare composts for their ability to slow the growth of R. solani. The assay also correlates well with suppression of other soilborne fungal pathogens that survive as saprophytes in soils such as Alternaria early blights, Fusarium wilt, Phytophthora root rot, and Pythium root rot.

Finished compost should be stable and mature, two terms that are often used interchangeably, so it can be safely packaged and transported, and not cause adverse effects during its end use4. Stability is a resistance to decomposition and is usually determined using indices of microbial activity. General measures of microbial respiration may measure compost stability but not necessarily disease suppression of an aggressive pathogen and saprophyte such as R. solani7. This study focused on maturity which infers the material is ready for a particular use, and, for horticultural purposes, is determined by plant germination and growth assays.
Extracts of the living composts suppressed R. solani growth significantly more than autoclaved samples (P &#8804; 0.0001). Autoclaving kills off microbial activity, pointing to a microbially mediated suppression. This confirms that Plate A in the method serves as a negative control for microbially mediated biological control (Figure 3).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58767/58767fig3.jpg" /> 
Figure 3: Effects of autoclaving on R. solani growth in vitro, as measured by percentage change in mycelial growth from control. A test material is &#39;suppressive&#39; to the fungus if the radius of mycelial growth is less than an autoclaved control. Illustrated are means &#177; 1 standard error. The error bar for the autoclaved control is too small to see. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58767/58767fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Growth of R. solani mycelium on compost extract was affected by compost method and raw material in the recipe of compost but not duration of maturation or curing6,8. Growth was reduced by vermicompost and windrow processes and recipes containing hardwood bark (Figure 4). Suppressiveness represents a reduction in growth of R. solani. 
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58767/58767fig4.jpg" /> 
Figure 4: Use of plate competition assay to compare the suppressiveness of compost products. Means &#177; 1 standard error of the change in R. solani growth on an autoclaved reference plate. Both controls and treatment comparisons were inoculated with virulent R. solani. Values below zero represent suppressiveness. Suppressiveness of R. solani was affected by compost A) method, B) duration of curing or maturation, and C) recipe. Processes compared were aerated static pile (ASP), windrow (W), vermicompost (V) and anaerobic digestion (AD). Recipe ingredients compared were food waste (F), poultry manure (P), food waste and poultry manure (FP), dairy manure (M), and hardwood bark (H). Contrasting letters above bars denote treatments that are significantly different. This figure has been modified from Neher et al.8&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58767/58767fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression at JoVE.com

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

Presented is a protocol for a plate competition assay to identify whether a specific compost is likely to contain bacteria and fungi that suppress growth of Rhizoctonia solani.

The goal was to develop and optimize a simple, affordable, and effective bioassay to detect disease suppressive ability of a specific compost against ...

a-plate-competition-assay-as-quick-preliminary-assessment-disease

Nanyang Technological University

Research

JoVE Journal

Biology

8.0K Views.  University of Vermont. Deze methode kan helpen bij het beantwoorden van belangrijke vragen met betrekking tot de biologische bestrijding van bodemgevoede plantenpathogenen. De gouden standaard voor het testen van materialen zoals compost is om een plant bioassay om te testen of het giftig is voor de planten. Dat duurt echter drie weken.Deze methode kan worden gedaan in ongeveer twee tot drie dagen. Het belangrijkste voordeel van deze techniek is dat het eenvoudig, betaalbaar en effectief is. Deze test is een...

Video: A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response following an infection. Despite efforts to identify unique surface marker on Tregs, the only unique feature is their ability to suppress the proliferation and function of effector T cells. While it is clear that only in vitro assays can be used in assessing human Treg function, this becomes problematic when assessing the results from cross-sectional studies where healthy cells and cells isolated from subjects with autoimmune diseases (like Type 1 Diabetes-T1D) need to be compared. There is a great variability among laboratories in the number and type of responder T cells, nature and strength of stimulation, Treg:responder ratios and the number and type of antigen-presenting cells (APC) used in human in vitro suppression assays. This variability makes comparison between studies measuring Treg function difficult. The Treg field needs a standardized suppression assay that will work well with both healthy subjects and those with autoimmune diseases. We have developed an in vitro suppression assay that shows very little intra-assay variability in the stimulation of T cells isolated from healthy volunteers compared to subjects with underlying autoimmune destruction of pancreatic &beta;-cells. The main goal of this piece is to describe an in vitro human suppression assay that allows comparison between different subject groups. Additionally, this assay has the potential to delineate a small loss in nTreg function and anticipate further loss in the future, thus identifying subjects who could benefit from preventive immunomodulatory therapy1. Below, we provide thorough description of the steps involved in this procedure. We hope to contribute to the standardization of the in vitro suppression assay used to measure Treg function. In addition, we offer this assay as a tool to recognize an early state of immune imbalance and a potential functional biomarker for T1D.

Human In Vitro Suppression as Screening Tool for the Recognition of an Early State of Immune Imbalance

Tregs are potent suppressors of the immune system. There is a lack of unique surface markers to define them, hence, definitions of Tregs are primarily functional. Here we describe an optimized in vitro assay capable of identifying immune imbalance in subjects at risk to develop T1D.

Regulatory T cells (Tregs) are critical mediators of immune tolerance to self-antigens. In addition, they are crucial regulators of the immune response ...

Immunology and Infection

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Cancer Research

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Phenotypic and Functional Analysis of Activated Regulatory T Cells Isolated from Chronic Lymphocytic Choriomeningitis Virus-infected Mice

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance and homeostasis maintenance by regulating the immune response. The primary role of Treg cells is to suppress the proliferation of effector T (Teff) cells and the production of cytokines such as IFN-&#947;, TNF-&#945;, and IL-2. It has been demonstrated that Treg cells&#39; ability to inhibit the function of Teff cells is enhanced during persistent pathogen infection and cancer development. To clarify the function of Treg cells under resting or inflamed conditions, a variety of in vitro suppression assays using mouse or human Treg cells have been devised. The main aim of this study is to develop a method to compare the differences in phenotype and suppressive function between resting and activated Treg cells. To isolate activated Treg cells, mice were infected with lymphocytic choriomeningitis virus (LCMV) clone 13 (CL13), a chronic strain of LCMV. Treg cells isolated from the spleen of LCMV CL13-infected mice exhibited both the activated phenotype and enhanced suppressive activity compared with resting Treg cells isolated from na&#239;ve mice. Here, we describe the basic protocol for ex vivo phenotype analysis to distinguish activated Treg cells from resting Treg cells. Furthermore, we describe a protocol for the measurement of the suppressive activity of fully activated Treg cells.

Here, we describe a protocol to analyze the phenotype of regulatory T (Treg) cells isolated from na&#239;ve and chronic lymphocytic choriomeningitis virus-infected mice. In addition, we provide a process to evaluate the suppressive activity of the Treg cells.

Regulatory T (Treg) cells, which express Foxp3 as a transcription factor, are subsets of CD4+ T cells. Treg cells play crucial roles in immune tolerance ...

Deferred Growth Inhibition Assay to Quantify the Effect of Bacteria-derived Antimicrobials on Competition

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential nutrient or produces antimicrobial compounds that its competitor is not resistant to. Here we describe a deferred growth inhibition assay, a method for assessing the ability of one bacterium to inhibit the growth of another through the production of antimicrobial compounds or through competition for nutrients. This technique has been used to investigate the correlation of nasal isolates with the exclusion of particular species from a community. This technique can also be used to screen for lantibiotic producers or potentially novel antimicrobials. The assay is performed by first culturing the test inhibitor-producing strain overnight on an agar plate, then spraying over the test competitor strain and incubating again. After incubation, the extent of inhibition can be measured quantitatively, through the size of the zone of clearing around the inhibitor-producing strain, and qualitatively, by assessing the clarity of the inhibition zone. Here we present the protocol for the deferred inhibition assay, describe ways to minimize variation between experiments, and define a clarity scale that can be used to qualitatively assess the degree of inhibition.

The deferred growth inhibition assay can be used to assess the competition effect by one bacterial isolate on another. Inhibition is quantified by measuring the zone of clearing around the inhibitor-producing isolate, or qualitatively assessed by determining the visible extent of inhibition.

Competitive exclusion can occur in microbial communities when, for example, an inhibitor-producing strain outcompetes its competitor for an essential ...

A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling contributes to the progression of many diseases. The ligand/receptor pair CXC chemokine ligand 12 (CXCL12)/CXC chemokine receptor 4 (CXCR4) has raised significant clinical interest, for instance as a potential target for the treatment of cancer and inflammatory diseases. Small molecules as well as therapeutic antibodies that specifically target CXCR4 and inhibit the receptor&#39;s function are therefore considered to be valuable pharmacological tools. Here, a flow cytometry-based cellular assay that allows identification of compounds (e.g., small molecules) that abrogate CXCL12 binding to CXCR4, is described. Essentially, the assay relies on the competition for receptor binding between a fixed amount of fluorescently labeled CXCL12, the natural chemokine agonist for CXCR4, and unlabeled compounds. Hence, the undesirable use of radioactively labeled probes is avoided in this assay. In addition, living cells are used as the source of receptor (CXCR4) instead of cell membrane preparations. This allows easy adaptation of the assay to a plate format, which increases the throughput. This assay has been shown to be a valuable generic drug discovery assay to identify CXCR4-targeting compounds. The protocol can likely be adapted to other GPCRs, at least if fluorescently labeled ligands are available or can be generated. Prior knowledge concerning the intracellular signaling pathways that are induced upon activation of these GPCRs, is not required.

A flow cytometry-based cellular binding assay is described that is primarily used as a screening tool to identify compounds that inhibit the binding of a fluorescently labeled CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4).

Pharmacological targeting of G protein-coupled receptors (GPCRs) is of great importance to human health, as dysfunctional GPCR-mediated signaling ...

Colony Formation Assay: Assessing the Efficacy of Anticancer Agents on Colony-Forming Lung Cancer Cells

Source: Lee, J. Y.et al, <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57490/preclinical-assessment-bioactivity-anticancer-coumarin-ot48-spheroids">Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts.</a>&#160;J. Vis. Exp.(2018).
This video describes the colony formation assay, which helps determine lung cancer cells&#39; ability to proliferate indefinitely and form individual colonies. We can analyze the effect of an anticancer agent on the colony-forming cells by analyzing the changes in the number and/or size of the resulting colonies.

Source: Lee, J. Y.et al, Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish ...

JoVE Encyclopedia of Experiments

Lung Cancer

In vitro study

ATP-Based Luciferase Viability Assay: A Homogenous Method to Evaluate the Growth-Inhibitory Potential of Test Agents on Tumor Organoids

Source: Higa, A. et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62668">High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids.</a> J. Vis. Exp. (2021)
In this video, we demonstrate a rapid, single-addition assay to assess cell viability in patient-derived organoids upon treatment with a test agent using intracellular ATP levels as a marker for cellular metabolic activity. The intracellular ATP levels are measured using the luciferase/luciferin reaction, where the emitted light intensity indicates the cellular viability and, in turn, the test agent&#39;s growth-inhibitory potency.

Source: Higa, A. et al. High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids. J. Vis. Exp. (2021)
In this video, we demonstrate a rapid, ...

Biological Techniques

Assay Techniques

Cell-based assays

Competitive Binding Assay to Identify Compounds Disrupting Receptor-Ligand Interactions

Source: Schoofs, G., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/57271">A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to CXC Chemokine Receptor 4.</a> J. Vis. Exp. (2018).
In this video, we describe a flow cytometry-based competitive binding assay to detect the interactions between the CXC Chemokine Receptor 4 (CXCR4) and its fluorescently-labeled natural ligand CXC Chemokine Ligand 12 (CXCL12), in the presence of a CXCR4-targeting small molecule. Incubating CXCR4-expressing cells with lower small molecule concentrations allows CXCR4-CXCL12 binding to some extent, which progressively declines upon a gradual increase in small molecule concentrations.

Source: Schoofs, G., et al. A Flow Cytometry-based Assay to Identify Compounds That Disrupt Binding of Fluorescently-labeled CXC Chemokine Ligand 12 to ...

A Plate Competition Assay As a Quick Preliminary Assessment of Disease Suppression

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