The development of this technique was supported by the funds from the Ministry of Science and Technology&#39;s National Key R&#38;D plan (2018YFA0902604), National Natural Science Foundation of China&#39;s Research Fund for International Young Scientists (22050410270) and Shenzhen Institutes of Advanced Technology External Funds (DWKF20190001). We would like to offer our sincere gratitude to Miss Chen Xinyi for her assistance in proofreading the document and laboratory management.

1. Preparation of gradient swarm plates
<ol>
	<li>Preparation of swarm medium 
	NOTE: See the discussion section for a brief comparison of different medium viscosities; 0.7% (w/v) agar concentration of swarm medium was used in this protocol.
	<ol>
		<li>Prepare Lysogeny broth (LB) powder with agar in two conical flasks; each flask contains 2 g of tryptone, 2 g of sodium chloride, 1 g of yeast extract, and 1.4 g of agar. Add double-distilled water (ddH2O) and stir the suspension using a magnetic stir bar. Adjust the final volume to 200 mL by adding additional ddH2O.</li>
		<li>Autoclave the solution at 121 &#176;C for 20 min. Use an air-permeable cap or bottle sealing film with an air vent. 
		NOTE: Agar will dissolve when heated in the autoclave.</li>
		<li>When the temperature drops to 65 &#176;C, mix the solution to ensure homogeneity, and transfer the medium to a 65 &#176;C incubator or water bath for short-term usage.</li>
	</ol>
	</li>
	<li>Preparation of bottom-layer swarm medium 
	NOTE: The bottom-layer medium is the mixture of swarm medium and inducer stock solutions. The formulation of inducer gradient swarm plates is shown in Table 1.
	<ol>
		<li>Prepare a 100 mM resveratrol stock solution by dissolving 114.12 mg of lyophilized resveratrol powder into 5 mL of dimethyl sulfoxide (DMSO), and store the solution at -20 &#176;C.</li>
		<li>Prepare a 20% (w/v) arabinose stock solution by dissolving 6 g of arabinose powder in 30 mL of ddH2O; wait for 10-15 min to allow the arabinose to dissolve; and store the solution at room temperature.</li>
		<li>Take out the medium from the 65 &#176;C incubator and place it at room temperature; allow the swarm medium to cool until the Erlenmeyer flask is cool enough to hold (~50 &#176;C). Do not place the swarm medium at room temperature for long periods, as this will cause the solidification of agar.</li>
		<li>Add the required volume of the inducer stock solution to the swarm medium at 50 &#176;C (Table 1). Use a pipette to dispense the inducer solution instead of pouring it. Gently swirl to mix the inducer with the swarm medium. 
		NOTE: This step is for inducers that cannot be autoclaved. Be careful not to introduce bubbles into the medium.</li>
	</ol>
	</li>
	<li>Preparation of double-layer gradient swarm plates 
	NOTE: The upper-layer medium is LB medium containing 0.7% (w/v) agar.
	<ol>
		<li>Label 13 x 13 cm square Petri dishes with inducer name and strains, and prop the dishes up over the edge of the lids (Figure 1B).</li>
		<li>Add 40 mL warm bottom-layer medium (50 &#176;C) using a 50 mL pipette or a 50 mL centrifuge tube. 
		NOTE: Alternatively, for 13 x 13 cm square Petri dishes, 40 mL bottom-layer and upper-layer medium is suitable; for 10 x 10 cm square Petri dishes, 25 mL bottom layer and upper-layer medium is suitable.</li>
		<li>Allow the bottom-layer medium to cure uncovered for 1 h inside a laminar flow hood. Do not disturb square Petri dishes while the medium solidifies. 
		NOTE: While curing the bottom layer, swarm medium not containing inducers should be maintained in a 65 &#176;C incubator or water bath.</li>
		<li>Once the bottom layer is completely solidified, remove the lids and lay the square Petri dishes inside a laminar flow hood.</li>
		<li>Add 40 mL of warm upper-layer medium (50 &#176;C) using a 50 mL pipette or 50 mL centrifuge tube. 
		NOTE: The upper-layer medium does not contain inducers.</li>
		<li>Cure the double-layer plates on the benchtop covered and undisturbed for 1 h. Store the prepared plates at 4 &#176;C for up to 24 h. 
		&#8203;NOTE: Longer curing times would reduce the moisture content and restrict swarming motility.</li>
	</ol>
	</li>
</ol>
2. Growth of E. coli K12 and P. aeruginosa PAO1
<ol>
	<li>Prepare 500 mL of LB medium by adding 5 g of tryptone, 5 g of NaCl, and 2.5 g of yeast extract into ddH2O, and top up the solution to 500 mL. Autoclave the solution on liquid cycle for 20 min at 121 &#176;C, and store it at 4 &#176;C.</li>
	<li>Prepare 100 mL of 1.5% (w/v) LB-agar medium by adding 1 g of tryptone, 1 g of NaCl, 0.5 g of yeast extract, and 1.5 g of agar into ddH2O and top up the solution to 100 mL. Autoclave the solution on liquid cycle for 20 min at 121 &#176;C. Transfer the medium to a 50 &#176;C water bath to prevent the agar from solidifying.</li>
	<li>When the LB-agar medium flask is comfortable to hold, add 20 mL of LB-agar medium into a Petri dish (10 cm in diameter) using a 25 mL pipette. Leave the plate at room temperature overnight, and store the LB-agar plate at 4 &#176;C.</li>
	<li>Take stock cultures stored at -80 &#176;C, streak E. coli K12, E. coli K12-YdeH, P. aeruginosa PAO1, and P. aeruginosa PAO1-YdeH strains on LB-agar Petri dishes using disposable inoculation loops. Incubate the Petri dishes inverted overnight at 37 &#176;C.</li>
	<li>Pick single colonies for different strains from the Petri dishes, inoculate each colony into 5 mL of LB medium, and incubate the culture at 37 &#176;C in a laboratory orbital shaker set at 220 rpm.</li>
	<li>When the culture density reaches OD600nm ~1.0, remove the culture from the shaker and place it at room temperature. Adjust the culture density to OD600nm = 1.0, as described in step 3.2.1.</li>
</ol>
3. Inoculation and incubation of gradient swarm plates
<ol>
	<li>Preparation of inoculation wells 
	NOTE: 3D printing cover models capable of generating wells separated by a standard distance can be used instead of the method described below (Supplemental Figure S1).
	<ol>
		<li>Mark the well positions on A4 paper shown in Figure 1C. Set three test concentrations in one square Petri dish with two or three replicates.</li>
		<li>Place the marked A4 paper under a solidified gradient plate. Push the broader side of a 100 &#181;L pipette tip into the semisolid medium surface at the marked position. Press the pipette tip until it reaches the bottom of the upper-layer medium.</li>
		<li>When the tip touches the bottom, apply no vertical force to the tip; gently rotate the tip to isolate the content of the cylindrical well.</li>
		<li>Horizontally move the pipette tips along a very small distance to allow airflow into the narrow place set aside. Press the tip with the index finger to block the gas flow inside the tip while holding the pipette using the thumb and middle finger.</li>
		<li>Pull the tip out vertically, keeping the well content in the tip while pulling it out. 
		NOTE: If the well content slips, apply slightly more pressure with the index finger to completely seal the tip.</li>
		<li>Repeat steps 3.1.2 to 3.1.5 in every marked position. Cover the swarm plate before inoculation.</li>
	</ol>
	</li>
	<li>Gradient plate inoculation and incubation
	<ol>
		<li>Adjust the overnight growth culture density to OD600nm = 1.0.</li>
		<li>Pipette 80 &#181;L of the overnight growth culture into every well. Do not spill the bacterial culture outside the wells.</li>
		<li>Wrap the plates with sealing film. For long-term observation (3-5 days), wrap the plates with sterile laboratory rubber tape. 
		NOTE: Rubber tape is less likely to break.</li>
		<li>Place a beaker filled with ddH2O in the incubator to maintain humidity inside the incubator. Incubate the gradient swarm plates at 37 &#176;C. 
		NOTE: Do not incubate the swarm plates upside-down; this will cause the bacterial culture to leak from the wells.</li>
		<li>Image the swarm plate immediately after inoculation, recording this as the 0 h time point.</li>
	</ol>
	</li>
</ol>
4. Imaging bacterial surface swarming
<ol>
	<li>Take the swarm plates out, one at a time, from the incubator every 12 h, holding the plate horizontally, and place them in the gel imaging system (see the Table of Materials). 
	NOTE: Do not leave fingerprints on the surface of plates; hold the side of the swarm plate with clean gloves.</li>
	<li>Select gel imaging mode; expose the swarm plate to white light; and adjust the focal length to give the clearest view of swarms. 
	NOTE: Use the same focal length for all plates in a given batch.</li>
	<li>Enhance the brightness of the swarms for clear observation by adjusting the exposure time to 300 ms. Adjust the threshold to minimize interference from the background light. 
	NOTE: Threshold is adjusted on the operating interface of the gel imaging system. Increase values on the left to minimize interference from background light; decrease values on the right to enhance the brightness of the swarms. In this protocol, the region is usually between 6,000 and&#160;50,000.</li>
	<li>Save the image file for further analysis. Record the imaging time, inducer type, gradient orientation, and strains in a .txt file.</li>
</ol>
5. Quantify the swarm area using ImageJ software
<ol>
	<li>Import the image file acquired using the gel imaging system.</li>
	<li>Set the scale bar using ImageJ software and apply it to all the images.
	<ol>
		<li>Create a line segment marking the length of the board by clicking on the line tool.</li>
		<li>Click Analyze | Set Scale to open the Set Scale window.</li>
		<li>Type the actual length in Known distance and Unit of length. 
		NOTE: Because 13 x 13 cm square Petri dishes were used in this work, the actual length is &#39;130&#39;, and the unit of length is &#39;mm.&#39;</li>
		<li>Check the Global box.</li>
		<li>Insert a scale bar by clicking Analyze | Tools | Scale Bar, type Width in mm, Height in pixels, Font size, and select Color, Background, and Location from the dropdown menu. Alternatively, choose Bold Text, Hide Text, Serif Font, and Overlay by ticking the checkboxes. 
		NOTE: The choice of those parameters is determined by users and the properties of the images. In this protocol, Width in mm was set to 25, Height in pixels to 20, Font size to 80, and the scale bar was placed in the lower right corner by selecting Location | Lower Right. Other parameters can be chosen by the user.</li>
	</ol>
	</li>
	<li>Click Process | Shadows to enhance the sharpness of the image, especially the boundaries (Supplemental Figure S2A). Click Process | Batch to process images. 
	NOTE: The purpose of this step is to provide more precise boundary demarcation.
	<ol>
		<li>Process an image as a reference by clicking on Process | Shadows | South.</li>
		<li>Click Process | Batch | Macro to open the Batch Process window. Look for the following commands displayed in the window: 
		run(&#34;South&#34;); 
		run(&#34;Save&#34;); 
		&#8203;close();</li>
		<li>Type the folder address of the original images and the output file address by clicking Process in the Batch Process window. 
		NOTE: It is recommended to export images with shadows to another folder and retain a copy of the original images.</li>
	</ol>
	</li>
	<li>Use Wand (tracing) tool to select swarms individually and adjust the tolerance (double-click Wand (tracing) tool) until the generated line fits the swarm boundary correctly (Supplemental Figure S2B). 
	NOTE: First, click Wand (tracing) tool and select a swarm on one image. If the boundaries have not been depicted correctly, double-click the Wand (tracing) tool to open the Wand Tool windows, where the Tolerance can be adjusted.</li>
	<li>Click on Analyze | Measure to export the area value.</li>
	<li>Repeat steps 5.1.1-5.1.5 until all swarms are measured, save the results to a .csv file for further analysis.</li>
</ol>

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The authors have no conflicts of interest to disclose.

Investigation of bacterial swarming motility on semisolid gradient plates can be a challenging task18,19,20, as it involves multiple factors such as substrate viscosity, humidity, and medium components. Among these factors, agar concentration plays a decisive role in determining microbial reversion to either swimming or swarming motility. As the agar concentration increases from 0.3% (w/v) to 1% (w/v), bacterial swimming motilit...

Bacterial swarming motility refers to the collective migration of bacterial cells across the surface of a substance. In addition to semisolid agar plates specially prepared in the laboratory1, this phenotype is also observed on some soft substrates such as animal tissues2, hydrated surfaces3, and plant roots4. While a semisolid surface is considered one of the fundamental conditions for bacterial swarming, some species also require an energy-rich medium to support their swarming motility5. Flagella rotation powers both swimming and swarming motility-swimming describes the unicellular motility within a liquid environment, whereas swarming is the synchronous movement of a microbial population across semisolid surfaces.
Substrate viscosity influences bacterial motility; studies of pathogenic microbes, such as Helicobacter pylori,&#160;have shown that the pathogen&#39;s motility changes depending on the mucin layer viscosity, which is influenced by environmental acidification in the human host6. To replicate these environments, earlier studies using agar concentration above 0.3% (w/v) restrict bacterial swimming motility to effect a gradual shift into surface swarming. The use of agar concentration above 1% (w/v) prevents the swarming motility of many species7. The colony patterns formed on the surface are diverse, including featureless mat8, bull&#39;s eye9, dendrites10, and vortex11.
Although the relevance of such patterns remains unclear, those patterns seem to be dependent on environmental and chemical cues12. Environmental cues cover different aspects, including temperature, salinity, light, and pH, whereas chemical cues include the presence of microbial quorum sensing molecules, biochemical byproducts, and nutrients. Autoinducer quorum sensing signaling molecules such as AHL (N-hexanoyl-L homoserine lactone) can impact surface swarming by regulating the production of surfactant13,14. Resveratrol, a phytoalexin compound, could restrict bacterial swarming motility15.
In the present work, we investigate the effect of gradient concentrations of resveratrol on wild-type Escherichia coli K12 strain and investigate arabinose-inducible swarming motility of engineered E. coli K12-YdeH and Pseudomonas aeruginosa PAO1-YdeH species. The production of the YdeH enzyme is induced by arabinose via the araBAD promoter, resulting in cellular c-di-GMP perturbation and affecting bacterial swarming motility16,17. This inducible swarming behavior is studied using arabinose gradient swarm plates with E. coli K12-YdeH and P. aeruginosa PAO1-YdeH strains.
The gradient swarm plates are prepared by successively solidifying double-layer medium (Figure 1B). The bottom layer comprises the medium added with the inducer, poured on one side of a propped-up Petri dish. Upon the solidification of the bottom layer, the Petri dish is returned to a flat surface, where the upper layer containing the medium without the inducer is added from the other side of the plate. After the swarm plates are completely solidified, isometrically arranged holding-wells are produced by boring holes on the swarm plates following a fixed layout (Figure 1C) or by imprinting the wells using a 3D printed model of the plate cover containing pegs during the medium curing process (Supplemental Figure S1). A gel imaging system is used to capture the swarming morphologies at different time points (Figure 2). Analysis of surface swarming using ImageJ software (Supplemental Figure S2) provides quantitative results of the surface swarming process (Figure 3).
Thus, we propose a simple method to test surface swarming motility within a concentration range of inducers. This method can be used to test multiple concentration responses of other inducers by mixing the inducer into the bottom-layer medium and can be applied to other motile species (e.g., Bacillus subtilis, Salmonella enterica, Proteus mirabilis, Yersinia enterocolitica). This approach could provide reliable qualitative and quantitative results for screening a single chemical inducer, and separate plates may be employed to evaluate more chemical inducers.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>V900500</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Solarbio</td>
			<td>A8180</td>
			<td>25 g, &#8805; 85% (GC)</td>
		</tr>
		<tr>
			<td>Centrifuge tube</td>
			<td>Corning</td>
			<td>430790</td>
			<td>15 mL</td>
		</tr>
		<tr>
			<td>Cryogenic vial</td>
			<td>Corning</td>
			<td>430488</td>
			<td>2 mL</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Aladdin</td>
			<td>D103272</td>
			<td>AR, &#62; 99% (GC)</td>
		</tr>
		<tr>
			<td>L(+)-Arabinose</td>
			<td>Aladdin</td>
			<td>A106195</td>
			<td>98% (GC), 500 g</td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Bkman</td>
			<td>B-SLPYM90-15</td>
			<td>Plastic Petri dishes,circular,90 mm x 15 mm</td>
		</tr>
		<tr>
			<td>Resveratrol</td>
			<td>Aladdin</td>
			<td>R107315</td>
			<td>99% (GC), 25 g</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Macklin</td>
			<td>S805275</td>
			<td>AR, 99.5% (GC), 500 g</td>
		</tr>
		<tr>
			<td>Square Petri dishes</td>
			<td>Bkman</td>
			<td>B-SLPYM130F</td>
			<td>Plastic Petri dishes, square, 13 mm x 13 mm</td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Thermo Scientific Oxoid</td>
			<td>LP0042</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Thermo Scientific Oxoid</td>
			<td>LP0021</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Biochemical incubator</td>
			<td>Blue pard</td>
			<td>LRH-70</td>
			<td></td>
		</tr>
		<tr>
			<td>Tanon 5200multi imaging system</td>
			<td>Tanon</td>
			<td>5200CE</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermostatic water bath</td>
			<td>Jinghong</td>
			<td>DK-S28</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantifying bacterial surface swarming motility on inducer gradient plates

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration&#160;of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time.
This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process.
The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers&#39; effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

The workflow consisting of the preparation of gradient swarm plates, inoculation, and incubation is shown in Figure 1B. To generate gradient swam plates, the bottom-layer medium is poured into propped-up dishes at ~4.3&#176; from the horizontal plane (Supplemental Figure S3), followed by pouring the upper-layer medium after the bottom layer is completely solidified. The composition of the double-layer medium is shown in Table 1. Then, bacterial culture cultu...

Watch this Scientific Journal Video about Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates at JoVE.com

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses.

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of ...

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Biochemistry

3.3K Views.  Southern University of Science and Technology. Here, we describe the use of inducer gradient plates to evaluate bacterial swarming motility while simultaneously obtaining multiple concentration responses. 

Video: Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Visualizing Surface T-Cell Receptor Dynamics Four-Dimensionally Using Lattice Light-Sheet Microscopy

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the structure-function relationship of these receptors in situ, we need to visualize and track them on the live cell surface with enough spatiotemporal resolution. Here we show how to use recently developed Lattice Light-Sheet Microscopy (LLSM) to image T-cell receptors (TCRs) four-dimensionally (4D, space and time) at the live cell membrane. T cells are one of the main effector cells of the adaptive immune system, and here we used T cells as an example to show that the signaling and function of these cells are driven by the dynamics and interactions of the TCRs. LLSM allows for 4D imaging with unprecedented spatiotemporal resolution. This microscopy technique therefore can be generally applied to a wide array of surface or intracellular molecules of different cells in biology.

The goal of this protocol is to show how to use Lattice Light-Sheet Microscopy to four-dimensionally visualize surface receptor dynamics in live cells. Here T cell receptors on CD4+ primary T cells are shown.

The signaling and function of a cell are dictated by the dynamic structures and interactions of its surface receptors. To truly understand the ...

A Planarian Motility Assay to Gauge the Biomodulating Properties of Natural Products

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and withdrawal properties of natural products is described. Experimental assays benefitting from unique aspects of planarian physiology have been applied to studies on wound healing, regeneration, and tumorigenesis. In addition, because planarians exhibit sensitivity to a variety of environmental stimuli and are capable of learning and developing conditioned responses, they can be used in behavioral studies examining learning and memory. Planarians possess a basic bilateral symmetry and a central nervous system that uses neurotransmitter systems amenable to studies examining the effects of neuromuscular biomodulators. Consequently, experimental systems monitoring planarian movement and motility have been developed to examine substance addiction and withdrawal. Because planarian motility offers the potential for a sensitive, easily standardized motility assay system to monitor the effect of stimuli, the planarian locomotor velocity (pLmV) test was adapted to monitor both stimulation and withdrawal behaviors by planarians through the determination of the number of grid lines crossed by the animals with time. Here, the technique and its application are demonstrated and explained.

Planarian motility is used to gauge the stimulant and withdrawal properties of natural products when compared to the movement of the animals in spring water alone.

A straightforward, controllable means of using the non-parasitic planarian, Dugesia tigrina, a free-living aquatic flatworm, to study the stimulant and ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Myosin-Specific Adaptations of In vitro Fluorescence Microscopy-Based Motility Assays

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic properties are adapted for the particular function they execute in cells. Myosin 5a processively walks on F-actin to transport melanosomes and vesicles in cells. Conversely, nonmuscle myosin 2b operates as a bipolar filament containing approximately 30 molecules. It moves F-actin of opposite polarity toward the center of the filament, where the myosin molecules work asynchronously to bind actin, impart a power stroke, and dissociate before repeating the cycle. Nonmuscle myosin 2b, along with its other nonmuscle myosin 2 isoforms, has roles that include&#160;cell adhesion, cytokinesis, and tension maintenance. The mechanochemistry of myosins can be studied by performing in vitro motility assays using purified proteins. In the gliding actin filament assay, the myosins are bound to a microscope coverslip surface and translocate fluorescently labeled F-actin, which can be tracked. In the single molecule/ensemble motility assay, however, F-actin is bound to a coverslip and the movement of fluorescently labeled myosin molecules on the F-actin is observed. In this report, the purification of recombinant myosin 5a from Sf9 cells using affinity chromatography is outlined. Following this, we outline two fluorescence microscopy-based assays: the gliding actin filament assay and the inverted motility assay. From these assays, parameters such as actin translocation velocities and single molecule run lengths and velocities can be extracted using the image analysis software. These techniques can also be applied to study the movement of single filaments of the nonmuscle myosin 2 isoforms, discussed herein in the context of nonmuscle myosin 2b. This workflow represents a protocol and a set of quantitative tools that can be used to study the single molecule and ensemble dynamics of nonmuscle myosins.

Presented here is a procedure to express and purify myosin 5a followed by a discussion of its characterization, using both ensemble and single molecule in vitro fluorescence microscopy-based assays, and how these methods can be modified for the characterization of nonmuscle myosin 2b.

Myosin proteins bind and interact with filamentous actin (F-actin) and are found in organisms across the phylogenetic tree. Their structure and enzymatic ...

Density Gradient Ultracentrifugation for Investigating Endocytic Recycling in Mammalian Cells

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma membrane and then transported to the early endosomes. The internalized proteins could be transited to the lysosome for degradation or recycled back to the plasma membrane. A robust endocytic recycling pathway is required to balance the removal of membrane materials from endocytosis. Various proteins are reported to regulate the pathway, including ADP-ribosylation factor 6 (ARF6). Density gradient ultracentrifugation is a classical method for cell fractionation. After the centrifugation, organelles are sedimented at their isopycnic surface. The fractions are collected and used for other downstream applications. Described here is a protocol to obtain a recycling endosome-containing fraction from transfected mammalian cells using density gradient ultracentrifugation. The isolated fractions were subjected to standard Western blotting for analyzing their protein contents. By employing this method, we identified that the plasma membrane targeting of engulfment and cell motility 1 (ELMO1), a Ras-related C3 botulinum toxin substrate 1 (Rac1) guanine nucleotide exchange factor, is through ARF6-mediated endocytic recycling.

This paper aims to present a protocol for preparing recycling endosomes from mammalian cells using sucrose density gradient ultracentrifugation.

Endosomal trafficking is an essential cellular process that regulates a broad range of biological events. Proteins are internalized from the plasma ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer&#39;s and Parkinson&#39;s diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...