This study was supported by a Korea Institute of Science and Technology intramural research grant (2E29563).

1. Preparation of P. aeruginosa PAO1 and Escherichia coli OP50 Culture
<ol>
	<li>Prepare 500 mL of sterilized Luria-Bertani (LB) medium (Table 1) and inoculate a single colony of P. aeruginosa into the medium. Incubate the culture for 14 to 15 h at 37 &#176;C with a shaking speed of 150 rpm.</li>
	<li>Equally distribute the bacterial culture into two 500 mL centrifuge tubes and centrifuge the tubes at 3,220 x g at 4 &#176;C for 30 min.</li>
	<li>Remove the supernatant until the volume is 50 mL (one-tenth of the initial volume) and resuspend the bacterial pellet.</li>
	<li>Store the concentrated bacterial culture at 4 &#176;C until use. The storage period of P. aeruginosa culture can be up to 1 month, but fresh culture is better for inducing intestinal damage. 
	NOTE: The protocol can be paused here. In addition to the whole live bacteria, the supernatant of the bacterial culture can be used to test the effects of the intestinal bacteria19.</li>
	<li>To prepare E. coli OP50, culture E. coli OP50 in DYT medium (Table 1) and then apply similar steps to those described above but decrease the volume to one-sixth of the original volume. For example, if the initial volume is 500 mL, after removing supernatant, the remaining volume is approximately 83 mL. 
	NOTE: The protocol can be paused here.</li>
</ol>
2. Preparation of Enterococcus faecalis KCTC 3206 Culture
<ol>
	<li>Prepare 500 mL of sterilized brain heart infusion (BHI) broth (Table 1).</li>
	<li>Inoculate one colony into 500 mL of BHI broth and incubate the culture for 14&#8211;15 h in a shaking incubator at 37 &#176;C and 150 rpm.</li>
	<li>Equally distribute the bacterial culture into two 500 mL centrifuge tubes and centrifuge the tubes at 3,220 x g at 4 &#176;C for 30 min.</li>
	<li>Remove the supernatant until the volume is 50 mL (one-tenth of the initial volume) and resuspend the pellet.</li>
	<li>Store the concentrated bacterial culture at 4 &#176;C until use. The fresh culture is better for testing the effects on the intestinal permeability. 
	NOTE: The protocol can be paused here.</li>
</ol>
3. Preparation of Heat-inactivated E. coli OP50 and Heat-inactivated P. aeruginosa PAO1 Cultures
<ol>
	<li>Culture and harvest the bacteria as described above (steps 1.1&#8211;1.3).</li>
	<li>Heat-inactivate the E. coli OP50 or P. aeruginosa PAO1 culture as described previously20,21,22. For heat inactivation, incubate the resuspended bacteria at 65 &#176;C (water bath) for 30 min.</li>
	<li>Cool the concentrated bacterial culture to room temperature and store it at 4 &#176;C until use. The storage period can be up to 1 month. 
	NOTE: The protocol can be paused here.</li>
</ol>
4. Preparation of Nematode Growth Medium (NGM) Plates for Testing the Effects of Different Bacteria on the Intestinal Permeability of C. elegans
<ol>
	<li>Place 1.25 g of peptone, 1.5 g of NaCl, 8.5 g of agar, a magnetic stirrer and 487.5 mL of distilled water in a 500 mL glass bottle (Table 1).</li>
	<li>Mix the mixture well, autoclave the mixture for 15 min at 121 &#176;C and cool the mixture to 55 &#176;C in a water bath for 30 min.</li>
	<li>Remove the medium from the water bath, add the components (0.5 mL of 1 M CaCl2, 0.5 mL of cholesterol, 0.5 mL of MgSO4, 12.5 mL of KPO4) (Table 1) to the NGM, and mix well. 
	NOTE: All the individual components must be autoclaved except for cholesterol (dissolved in absolute ethanol), and every experimental step must be carried out on a clean bench.</li>
	<li>Distribute 20 mL of NGM to each 90 x 15 mm Petri dish and allow the agar to solidify on a clean bench at room temperature (approximately 20 &#176;C). The NGM plates can be stored for up to one month at 4 &#176;C. 
	NOTE: The protocol can be paused here. The NGM plates used for FITC-dextran staining were prepared by the same procedure (from steps 4.1&#8211;4.3). 10 mL of NGM was distributed to each 60 x 15 mm Petri dish and allowed to solidify on a clean bench at room temperature (approximately 20 &#176;C).</li>
	<li>Remove the bacterial culture from the 4 &#176;C refrigerator and vortex the culture thoroughly before spreading the culture onto the NGM plates.</li>
	<li>Add a total of 800 &#181;L of the bacterial culture to each fresh NGM plate, and allow the plates to dry in a 20 &#176;C incubator overnight. 
	NOTE: For the first experiment (Figure 1), two NGM plates with E. coli OP50, one NGM plate with P. aeruginosa PAO1, and one NGM plate with E. faecalis KCTC3206 were prepared. For the second experiment (Figure 2), two NGM plates with live E. coli OP50, one NGM plate with live P. aeruginosa PAO1, and one NGM plate with heat-inactivated P. aeruginosa PAO1 were prepared.</li>
</ol>
5. Preparation of NGM Plates for Testing the Effects of a Chemical (DIM) on the Intestinal Permeability of C. elegans Fed with P. aeruginosa
<ol>
	<li>Add 0.5 g of peptone, 0.6 g of NaCl, 195 mL of distilled water, a magnetic stirrer, and 6.8 g of agar to a 500 mL glass bottle (NGM agar).</li>
	<li>Add 0.5 g of peptone, 0.6 g of NaCl, 195 mL of distilled water, and a magnetic stirrer without agar to another 500 mL glass bottle (NGM broth).</li>
	<li>Autoclave the two bottles (from steps 5.1&#8211;5.2), one empty 100 mL glass bottle, and one empty 500 mL glass bottle for 15 min at 121 &#176;C. Then, allow the medium containing bottles to cool to 55 &#176;C in the water bath for 30 min. Keep the agar medium in the water bath and remove the bottle containing broth (from step 5.2) for the next step.</li>
	<li>Add the additive chemicals to the NGM broth: 0.4 mL of 1 M CaCl2, 0.4 mL of cholesterol in ethanol (mL), 0.4 mL of 1 M MgSO4 and 10 mL of 1 M KPO4 (all of these components must be sterilized apart from the cholesterol in ethanol). Then, stir the mixture thoroughly with a magnetic stirrer at 55 &#176;C.</li>
	<li>Label the autoclaved empty 500 mL glass bottle with dimethyl sulfoxide (DMSO) and the autoclaved empty 100 mL glass bottle with DIM.</li>
	<li>Transfer 50 mL of NGM broth into the DIM-labeled 100 mL bottle. Add 500 &#181;L of 20 mM DIM stock into the bottle and mix well. 
	NOTE: DIM is dissolved in DMSO (20 mM DIM stock).</li>
	<li>Quickly remove the NGM agar medium from the water bath, add 50 mL of the NGM agar medium into the DIM-labeled bottle and mix thoroughly.</li>
	<li>Place a 20 mL aliquot of DIM-containing NGM medium into each 90 mm x 15 mm Petri dish. Approximately five DIM-containing NGM plates can be made from this step. 
	NOTE: The final concentration of DIM in each NGM plate will be 100 &#181;M.</li>
	<li>To prepare the DMSO-containing NGM plate, transfer 150 mL of NGM broth into the DMSO-labeled 500 mL bottle. Add 1.5 mL of DMSO to the bottle and mix well.</li>
	<li>Add 150 mL of the NGM agar medium to the DMSO-labeled bottle and mix thoroughly.</li>
	<li>Place a 20 mL aliquot of DMSO-containing NGM medium in each 90 mm x 15 mm Petri dish. Approximately fifteen DMSO-containing NGM plates can be made from this step. 
	NOTE: The final concentration of DMSO in each NGM plate will be 0.5%.</li>
	<li>Solidify the plates at room temperature for at least 3 h and store them at 4 &#176;C until use. 
	NOTE: The protocol can be paused here.</li>
	<li>Remove the E. coli OP50 or P. aeruginosa PAO1 culture from the 4 &#176;C refrigerator (from step 1.4) and vortex the culture properly before spreading onto the NGM plates.</li>
	<li>Put 800 &#181;L of the E. coli OP50 or P. aeruginosa PAO1 bacterial culture onto each fresh NGM agar plate and allow the plates to dry in a 20 &#176;C incubator overnight. 
	NOTE: The protocol can be paused here. For the third experiment (Figure 3), two DMSO-containing NGM plates coated with live E. coli OP50, one DMSO-containing NGM plate coated with live P. aeruginosa PAO1, and one DIM-containing NGM plate coated with live P. aeruginosa PAO1 were prepared.</li>
</ol>
6. Preparation of Age-synchronized C. elegans
<ol>
	<li>Grow worms on a solid NGM plate and feed them with E. coli OP50 until the desired population is reached.</li>
	<li>Synchronize the eggs by using either the timed egg laying method or the bleaching solution treatment as described previously20,23,24.</li>
</ol>
7. Treatment of Bacteria or DIM and FITC-dextran Feeding
<ol>
	<li>Incubate the age-synchronized eggs at 20 &#176;C for 64 h on NGM plates supplemented with live E. coli OP50 as food.</li>
	<li>Wash the age-synchronized L4 larva with S-buffer and transfer them (more than approximately 500 worms) to the treatment NGM plates containing different bacteria and chemicals (described in NOTE following step 4.6 and 5.14,). Incubate them at 20 &#176;C for 48 h. 
	NOTE: The treatment time can range from 24 to 72 h, according to the bacteria and chemicals used. The therapeutic or preventive effect of DIM can also be evaluated by pretreating the worms with pathogens and then treating the worms with DIM (the therapeutic effect) or by pretreating the worms with DIM and then treating with pathogens (the preventive effect)19.</li>
	<li>To prepare the FITC-dextran-supplemented plates, mix 2 mL of heat-inactivated E. coli OP50 with 4 mg of FITC-dextran. Then, divide 100 &#181;L of the FITC-dextran and E. coli OP50 mixture into twenty fresh NGM agar plates (60 mm x 15 mm) and allow the plates to dry for 1 h on a clean bench. 
	NOTE: The final concentration of FITC-dextran in each NGM plate will be 20 &#956;g/mL.</li>
	<li>Prepare five E. coli OP50-containing NGM plates without FITC-dextran for the vehicle control treatment. For this purpose, divide 100 &#181;L heat-inactivated E. coli OP50 to each fresh NGM agar plates (60 mm x 15 mm) and allow the plates to dry for 1 h on a clean bench. 
	NOTE: For each independent experiment, fifteen FITC-dextran-supplemented NGM plates and five NGM plates without FITC-dextran are required.</li>
	<li>After 48 h of treatment (from step 7.2), wash the worms with S-buffer, transfer the worms to the FITC-dextran supplemented plates and the NGM plates without FITC-dextran, and incubate the plates overnight (14&#8211;15 h). 
	NOTE: For each treatment group, 5 replicates of FITC-dextran staining (or vehicle control feeding) are required.</li>
	<li>Wash the worms with S-buffer and allow them to crawl in the fresh NGM agar plate for 1 h. 
	NOTE: For each independent experiment, a total of 20 fresh NGM plates are needed. In this step, you can use the NGM plate supplemented without or with E. coli OP50.</li>
	<li>Add 50 &#181;L of 4% formaldehyde solution to each well of a black 96-well flat-bottom plate. Transfer approximately 50 worms from each NGM plate into each well for fluorescence measurements. After 1 to 2 min, thoroughly remove all the formaldehyde from each well and add 100 &#181;L of mounting medium to coat the wells. 
	NOTE: Formaldehyde solution (4%) is used to immobilize and fix the worms. For each treatment group, 5 wells (5 replicates) are used for image analysis.</li>
</ol>
8. Imaging C. elegans with the Operetta Imaging System and Determination of Intestinal Permeability by Measuring the FITC-dextran Fluorescence Uptake
NOTE: Fluorescent stereomicroscopy can be used for image analysis instead of the Operetta system.
<ol>
	<li>Capture fluorescence images and measure the fluorescence intensity using the Operetta High-Content Imaging System and analyze the images with Harmony software.</li>
	<li>In the Harmony software, press the icon Open lid to open the lid and to put the plate into the machine.</li>
	<li>Set up the parameters.</li>
	<li>Click Set up, select the plate type (96-well corning flat-bottom) and add the channels (bright-field and EGFP channels).</li>
	<li>Adjust the layout. Go to the Layout selection, and then select Track and adjust the parameters (first picture at 1 &#181;m, number of planes are 10, distance is 1 &#181;m).</li>
	<li>Select one of the treatment wells and one capture field and press Test to check whether the pictures are satisfactory in the Run experiment section.</li>
	<li>If the pictures are satisfactory, return to the Set up section and press the Reset icon at the end of the screen. Then, select all the target wells and a suitable number of capture fields.</li>
	<li>Go to Run experiment again and enter the plate name, then press Start to begin processing.</li>
	<li>To measure the intensity of the fluorescence, go to the image analysis section and input the image. Find the cell by choosing the EGFP channel and method B. Adjust the common threshold to 0.5, area to &#62;200 &#181;m2, split factor to 3.0, individual threshold to 0.18 and contrast to more than 0.18. Then, calculate the intensity properties and choose the mean as the output. Press the Apply icon to save the setup.</li>
	<li>Go to the Evaluation section to measure the intensity by obtaining a heatmap and data table. Set the Readout parameter to Cells &#8212; Intensity Cell EGFP Mean &#8212; Mean per Well and start the evaluation. 
	NOTE: The protocol can be paused here.</li>
	<li>To extract the data from Operetta, click the Setting button and choose Data management.</li>
	<li>Choose Write archive, and then open the browse and select the file.</li>
	<li>To select the file, click the small + signal at the left corner, and then click Measurement and choose Plate name.</li>
	<li>Select the file and click OK.</li>
	<li>Select the path to save the file by clicking the Browse signal at the active path.</li>
	<li>Click Start to save the data file. 
	NOTE: The protocol can be paused here.</li>
</ol>
9. Statistical Analysis of the FITC-dextran Fluorescence of C. elegans
<ol>
	<li>Import the data and calculate the mean and standard deviation (SD) using a statistics software.</li>
	<li>Analyze the significant difference with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.</li>
</ol>

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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature-dependent+inactivating+factor+of+Pseudomonas+aeruginosa+exotoxin+A.">Temperature-dependent inactivating factor of Pseudomonas aeruginosa exotoxin A.</a> Infection and Immunity. 13 (5), 1467-1472 (1976).</li><li>Horii, T., Muramatsu, H., Monji, A., Miyagishima, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+exotoxin+A,+peptidoglycan+and+endotoxin+after+exposure+of+clinical+Pseudomonas+aeruginosa+isolates+to+carbapenems+in+vitro.">Release of exotoxin A, peptidoglycan and endotoxin after exposure of clinical Pseudomonas aeruginosa isolates to carbapenems in vitro.</a> Chemotherapy. 51 (6), 324-331 (2005).</li><li>Kirikae, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+characterization+of+endotoxins+released+from+antibiotic-treated+Pseudomonas+aeruginosa+and+Escherichia+coli.">Biological characterization of endotoxins released from antibiotic-treated Pseudomonas aeruginosa and Escherichia coli.</a> Antimicrobial Agents and Chemotherapy. 42 (5), 1015-1021 (1998).</li><li>Morlon-Guyot, J., Mere, J., Bonhoure, A., Beaumelle, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+Pseudomonas+aeruginosa+exotoxin+A+is+dispensable+for+cell+intoxication.">Processing of Pseudomonas aeruginosa exotoxin A is dispensable for cell intoxication.</a> Infection and Immunity. 77 (7), 3090-3099 (2009).</li><li>Kim, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3,3'-diindolylmethane+attenuates+colonic+inflammation+and+tumorigenesis+in+mice.">3,3'-diindolylmethane attenuates colonic inflammation and tumorigenesis in mice.</a> Inflammatory Bowel Diseases. 15 (8), 1164-1173 (2009).</li><li>Kanmani, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probiotics+and+its+functionally+valuable+products-a+review.">Probiotics and its functionally valuable products-a review.</a> Critical Reviews in Food Science and Nutrition. 53 (6), 641-658 (2013).</li><li>Nguyen, M. T., Gotz, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoproteins+of+Gram-Positive+Bacteria:+Key+Players+in+the+Immune+Response+and+Virulence.">Lipoproteins of Gram-Positive Bacteria: Key Players in the Immune Response and Virulence.</a> Microbiology and Molecular Biology Reviews. 80 (3), 891-903 (2016).</li></ol>

The authors have nothing to disclose.

By utilizing this new method for determining gut permeability in C. elegans, which combines automated fluorescence microscopy and quantitative image analysis, the differences caused by intestinal microorganisms or chemicals can be determined in vivo, specifically in the C. elegans intestine. This protocol is useful for gut permeability investigations and applicable to many tasks, such as reactive oxygen species (ROS) determination under stress conditions and morphological examinations, due to its conven...

Intestinal permeability is considered as one of the main barriers related to the intestinal microbiota and mucosal immunity and is likely to be affected by several factors, such as gut microbiota modifications, epithelial impairment, or mucus layer alterations1. Recent papers have reported effective protocols to measure the intestinal permeability of cultured human intestinal cells by analyzing the fluorescence flux rates across the intestinal cell layer2, but fewer research papers present a suitable procedure for measuring the gut permeability in nematodes, particularly in C. elegans, by using FITC-dextran staining.
There are two representative protocols for measuring the gut permeability in C. elegans using Nile red3 and erioglaucine disodium (or the Smurf assay)4,5. In this protocol, we used FITC-dextran (average molecular weight 10,000), which has a much higher molecular weight than Nile red (MW = 318.37) and erioglaucine disodium (MW = 792.85). FITC-dextran is more similar than Nile red or erioglaucine disodium dyes to actual macromolecular nutrients such as carbohydrates, which are absorbed through the intestinal layer. The intestinal permeability of C. elegans fed with erioglaucine disodium (blue Smurf dye) can be easily evaluated without fluorescence microscopy. However, in the Smurf assay, quantitative analysis of intestinal permeability is difficult due to the lack of standardization and should be evaluated manually4,5. In the case of the Nile red assay, Nile red also stains lipid droplets in cells, which may interfere with the exact determination of gut permeability in C. elegans6. The present protocols enable rapid and precise quantitative analysis of intestinal permeability in C. elegans treated with various intestinal bacteria and chemicals while avoiding unspecific lipid staining.
C. elegans is a typical model in biological fields due to its affordable price, easy manipulation, limited animal ethics issues, and short lifespan, which is beneficial for rapid experimentation7. In particular, after the entire C. elegans genome was published, nearly 40% of genes in the C. elegans genome were found to be orthologous to genes that cause human diseases8. Moreover, the transparent body allows observation inside the organism, which is advantageous for researching cellular events and for fluorescence applications in cell biology, for example, stem cell staining with DAPI or immunohistochemistry9. C. elegans is often used as an experimental animal to study the interaction between the gut microbiota and the host; in addition, C. elegans is used to screen health-promoting probiotic bacteria10,11,12 as well as dietary chemicals promoting intestinal health13,14.
Pseudomonas aeruginosa and Enterococcus faecalis are well-known gut bacteria that negatively affect the gastrointestinal system, especially the colonic epithelial cells of the intestinal tract15,16. Therefore, measuring the gut permeability triggered by these bacteria is necessary for the screening and development of new drugs that can recover and reduce the damage caused by bacterial inflammation and infection. In this protocol, we tested the effects of these intestinal bacteria on the intestinal permeability of C. elegans.
We also report an optimized protocol for testing chemicals on the intestinal permeability of C. elegans. For this purpose, we used 3,3'-diindolylmethane (DIM) as a model chemical because DIM is a bioactive metabolite compound derived from indole-3-carbinol, which is present in Brassica food plants, and has been reported to have therapeutic effects on IBD in mice17,18. In addition, we recently discovered that DIM improves intestinal permeability dysfunction in both cultured human intestinal cells as well as the model nematode C. elegans19.
In this study, we used three different experimental conditions. First, we measured the effects of the different bacteria, P. aeruginosa and E. faecalis, on intestinal permeability (Figure 1). Second, we measured the effects of live and heat-inactivated P. aeruginosa on intestinal permeability (Figure 2). Third, we measured the effects of DIM (a model chemical) on the intestinal permeability of C. elegans fed with P. aeruginosa (Figure 3).
The objective of this study was to develop optimized protocols that measure the intestinal permeability of C. elegans, which is changed by treatment with various intestinal bacteria as well as with chemicals.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >3,3&#8217;-diindolylmethane&#160;</td>
			<td >Sigma</td>
			<td >D9568</td>
			<td ></td>
		</tr>
		<tr >
			<td >90&#215;15 mm Petri dishes</td>
			<td >SPL Life Sciences, South Korea</td>
			<td >10090</td>
			<td></td>
		</tr>
		<tr >
			<td >60&#215;15 mm Petri dishes</td>
			<td >SPL Life Sciences, South Korea</td>
			<td >10060</td>
			<td></td>
		</tr>
		<tr >
			<td >Bactor Agar</td>
			<td >Beckton Dickinson</td>
			<td >REF. 214010</td>
			<td></td>
		</tr>
		<tr >
			<td >Formaldehyde solution&#160;</td>
			<td >Sigma</td>
			<td >F1635</td>
			<td></td>
		</tr>
		<tr >
			<td >Brain Heart Infusion (BHI)&#160;</td>
			<td >Becton Dickinson</td>
			<td >REF. 237500</td>
			<td></td>
		</tr>
		<tr >
			<td >Caenorhabditis elegans N2</td>
			<td >Caenorhabditis Genetics Center (CGC)</td>
			<td ></td>
			<td>Wild type&#160;</td>
		</tr>
		<tr >
			<td >Cholesterol</td>
			<td >Sigma</td>
			<td >C3045</td>
			<td></td>
		</tr>
		<tr >
			<td >Costa Assay Plate, 96 Well Black With Clear Flat Bottom Non-treated, No Lid Polystyrene</td>
			<td >Corning Incorporated</td>
			<td >REF. 3631</td>
			<td></td>
		</tr>
		<tr >
			<td >Dimethyl sulfoxide</td>
			<td >Sigma</td>
			<td >D2650</td>
			<td></td>
		</tr>
		<tr >
			<td >Enterococcus faecalis KCTC 3206</td>
			<td >Korean Collection for Type Culture</td>
			<td >KCTC NO. 3206</td>
			<td>Falcutative anaerobic</td>
		</tr>
		<tr >
			<td >Escherichia coli OP50</td>
			<td >Caenorhabditis Genetics Center (CGC)</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescein isothiocyanate - dextran</td>
			<td >Sigma</td>
			<td >FD10S</td>
			<td></td>
		</tr>
		<tr >
			<td >Harmony software&#160;</td>
			<td >PerkinElmer</td>
			<td ></td>
			<td>verson 3.5</td>
		</tr>
		<tr >
			<td >Luria-Bertani LB medium</td>
			<td >Merck</td>
			<td >VM743185 626&#160; 1.10285.5000</td>
			<td></td>
		</tr>
		<tr >
			<td >Magnesium sulfate heptahydrate&#160;</td>
			<td >Fisher Bioreagents</td>
			<td >BP2213-1</td>
			<td></td>
		</tr>
		<tr >
			<td >Fluoromount aqueous mounting medium</td>
			<td >Sigma</td>
			<td >F4680</td>
			<td></td>
		</tr>
		<tr >
			<td >Operetta CLS High-Content Analysis System</td>
			<td >PerkinElmer</td>
			<td>&#160;HH16000000</td>
			<td></td>
		</tr>
		<tr >
			<td >Peptone</td>
			<td >Merck</td>
			<td >EMD 1.07213.1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Pseudomonas aeruginosa PA01</td>
			<td >Korean Collection for Type Culture</td>
			<td >KCTC NO. 1637</td>
			<td></td>
		</tr>
		<tr >
			<td >Sodium Chloride</td>
			<td >Fisher Bioreagents</td>
			<td >BP358-1</td>
			<td></td>
		</tr>
		<tr >
			<td >Stereo Microscope</td>
			<td >Nikon, Japan</td>
			<td >SMZ800N</td>
			<td></td>
		</tr>
		<tr >
			<td >Yeast extract</td>
			<td >Becton Dickinson</td>
			<td >REF. 212750</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring the effects of bacteria and chemicals on the intestinal permeability of caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a nonmammalian animal model that is widely used as an assay system due to its short lifespan, transparency, cost-effectiveness, and lack of animal ethics issues. In this study, a method was developed to investigate the effects of different bacteria and 3,3'-diindolylmethane (DIM) on the intestinal permeability of C. elegans with a high-throughput image analysis system. The worms were infected with different gut bacteria or cotreated with DIM for 48 h and fed with fluorescein isothiocyanate (FITC)-dextran overnight. Then, the intestinal permeability was examined by comparing the fluorescence images and the fluorescence intensity inside the worm bodies. This method may also have the potential to identify probiotic and pathogenic intestinal bacteria that affect intestinal permeability in the animal model and is effective for examining the effects of harmful or health-promoting chemicals on intestinal permeability and intestinal health. However, this protocol also has some considerable limitations at the genetic level, especially for determining which genes are altered to control illness, because this method is mostly used for phenotypic determination. In addition, this method is limited to determining exactly which pathogenic substrates cause inflammation or increase the permeability of the worms' intestines during infection. Therefore, further in-depth studies, including investigation of the molecular genetic mechanism using mutant bacteria and nematodes as well as chemical component analysis of bacteria, are required to fully evaluate the function of bacteria and chemicals in determining intestinal permeability.

After incubation with P. aeruginosa PAO1, C. elegans showed a significant increase in FITC-dextran fluorescence in the worm body compared to the fluorescence shown after incubation with the other two bacterial strains (Figure 1). The fluorescence intensities of worms fed with E. coli OP50, P. aeruginosa PAO1, and E. faecalis KCTC3206 were 100.0 &#177; 6.6, 369.7 &#177; 38.9, and 105.6 &#177; 10.6%, respectively. The data emphasize that P. aeru...

Watch this Scientific Journal Video about Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans at JoVE.com

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

In living organisms, intestinal hyperpermeability is a serious symptom that leads to many inflammatory bowel diseases (IBDs). Caenorhabditis elegans is a ...

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13.8K Views.  Korea Institute of Science and Technology. This protocol describes how to measure intestinal permeability of Caenorhabditis elegans. This method is helpful for basic biological research on intestinal health related to the interaction between intestinal bacteria and their host and for screening to identify probiotic and chemical agents to cure leaky gut syndrome and inflammatory bowel diseases.

Video: Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans

Measuring Frailty in HIV-infected Individuals. Identification of Frail Patients is the First Step to Amelioration and Reversal of Frailty

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased reserve and resistance to stressors increases in incidence with increasing age. In the elderly, frailty may pursue a step-wise loss of function from non-frail to pre-frail to frail. We studied frailty in HIV-infected patients and found that ~20% are frail using the Fried phenotype using stringent criteria developed for the elderly1,2. In HIV infection the syndrome occurs at a younger age.
HIV patients were checked for 1) unintentional weight loss; 2) slowness as determined by walking speed; 3) weakness as measured by a grip dynamometer; 4) exhaustion by responses to a depression scale; and 5) low physical activity was determined by assessing kilocalories expended in a week's time. Pre-frailty was present with any two of five criteria and frailty was present if any three of the five criteria were abnormal.
The tests take approximately 10-15 min to complete and they can be performed by medical assistants during routine clinic visits. Test results are scored by referring to standard tables. Understanding which of the five components contribute to frailty in an individual patient can allow the clinician to address relevant underlying problems, many of which are not evident in routine HIV clinic visits.

Frailty syndrome is commonly seen in the aged and reflects multi-system physiological change. However, with reduced functional reserve and resilience frailty is also known to be common in the HIV infected population. This study outlined an easily administered screening test to identify HIV patients with frailty. When significant components of frailty are identified, clinicians will be able to focus on amelioration of the problem and promote reversion to the pre-frail state.

A simple, validated protocol consisting of a battery of tests is available to identify elderly patients with frailty syndrome. This syndrome of decreased ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using &#34;intestinal sacs&#34; are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (Papp) of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

This protocol describes the use of excised intestinal tissue preparations or "intestinal sacs" as an ex vivo model of intestinal barrier function. This model may be used to assess integrity of both the epithelial barrier and the mucous gel layer at specific intestinal sites in animal models of digestive disease.

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Analyzing Beneficial Effects of Nutritional Supplements on Intestinal Epithelial Barrier Functions During Experimental Colitis

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause severe problems, such as abdominal cramping, bloody diarrhea, and weight loss, in affected individuals. Unfortunately, there is no cure yet, and treatments only aim to alleviate symptoms. Current treatments include anti-inflammatory and immunosuppressive drugs that may cause severe side effects. This warrants the search for alternative treatment options, such as nutritional supplements, that do not cause side effects. Before their application in clinical studies, such compounds must be rigorously tested for effectiveness and security in animal models. A reliable experimental model is the dextran sulfate sodium (DSS) colitis model in mice, which reproduces many of the clinical signs of ulcerative colitis in humans. We recently applied this model to test the beneficial effects of a nutritional supplement containing vitamins C and E, L-arginine, and &#969;3-polyunsaturated fatty acids (PUFA). We analyzed various disease parameters and found that this supplement was able to ameliorate edema formation, tissue damage, leukocyte infiltration, oxidative stress, and the production of pro-inflammatory cytokines, leading to an overall improvement in the disease activity index. In this article, we explain in detail the correct application of nutritional supplements using the DSS colitis model in C57Bl/6 mice, as well as how disease parameters such as histology, oxidative stress, and inflammation are assessed. Analyzing the beneficial effects of different diet supplements may then eventually open new avenues for the development of alternative treatment strategies that alleviate IBD symptoms and/or that prolong the phases of remission without causing severe side effects.

Current treatments of inflammatory bowel diseases (IBD) aim at alleviating disease symptoms but may cause severe side effects. Thus, alternative strategies are being investigated in animal colitis models. Here, we explain how the beneficial effects of diet supplements on clinical IBD signs are analyzed in such a colitis model.

Inflammatory bowel diseases (IBD), including Crohn&#39;s disease and ulcerative colitis, are chronic relapsing disorders of the intestines. They cause ...

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A non-mammalian soil round worm, Caenorhabditis elegans, is a valuable model organism for toxicology studies due to its convenience and lack of animal ethics issues compared with mammalian animal systems. In this protocol, a detailed procedure of toxicological evaluation of chemicals in C. elegans is described. A clinical anticancer drug, etoposide, which targets human topoisomerase II and inhibits DNA replication of human cancer cells, was selected as a model testing chemical. Age-synchronized C. elegans eggs were exposed to either dimethyl sulfoxide (DMSO) or etoposide, and then the growth of C. elegans was monitored every day for 4 days by the stereo microscope observation. The total number of eggs laid from C. elegans treated with DMSO or etoposide was also counted by using the stereo microscope. Etoposide treatment significantly affected the growth and reproduction of C. elegans. By comparison of the total number of eggs laid from worms with different treatment periods of chemicals, it can be decided that the reproductive toxicity of chemicals on C. elegans reproduction is reversible or irreversible. These protocols may be helpful for both the development of various drugs and risk assessment of environmental toxicants.

Measuring the Effect of Chemicals on the Growth and Reproduction of Caenorhabditis elegans

A basic protocol to evaluate the toxicity of chemicals in a model animal, Caenorhabditis elegans, is described. The method is convenient and useful for the development of pharmaceuticals as well as for the risk assessment of various environmental pollutants.

Toxicological evaluation is crucial for understanding the effects of chemicals on living organisms in basic and applied biological science fields. A ...

A Murine Model of Fetal Exposure to Maternal Inflammation to Study the Effects of Acute Chorioamnionitis on Newborn Intestinal Development

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing enterocolitis (NEC). However, a mechanistic link between these two conditions remains yet to be discovered. We have adopted a murine model of chorioamnionitis involving lipopolysaccharide (LPS)-induced fetal exposure to maternal inflammation (FEMI). This model of FEMI induces a sterile maternal, placental, and fetal inflammatory cascade, which is also present in many cases of clinical chorioamnionitis. Although models exist that utilize live bacteria and more accurately mimic the pathophysiology of an ascending infection resulting in chorioamnionitis, these methods may cause indirect effects on development of the immature intestinal tract and the associated developing microbiome. Using this protocol, we have demonstrated that LPS-induced FEMI results in a dose-dependent increase in pregnancy loss and preterm birth, as well as disruption of normal intestinal development in offspring. Further, we have demonstrated that FEMI significantly increases intestinal injury and serum cytokines in offspring, while simultaneously decreasing goblet and Paneth cells, both of which provide a first line of innate immunity against intestinal inflammation. Although a similar model of LPS-induced FEMI has been used to model the association between chorioamnionitis and subsequent abnormalities of the central nervous system, to our knowledge, this protocol is the first to attempt to elucidate a mechanistic link between chorioamnionitis and later perturbations in intestinal development as a potential link between chorioamnionitis and NEC.

We developed a model of chorioamnionitis to simulate fetal exposure to maternal inflammation (FEMI) without complications of live organisms to examine the effects of FEMI on development of the offspring&#8217;s intestinal tract. This allows for study of mechanistic causes for development of intestinal injury following chorioamnionitis.

Chorioamnionitis is a common precipitant of preterm birth and is associated with many of the morbidities of prematurity, including necrotizing ...

Isolation of Murine Intestinal Mesenchyme Resulting in a High Yield of Telocytes

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, fibroblasts, myofibroblasts, smooth muscle cells, immune cells, and the recently identified cell type, telocytes. Telocytes are unique mesenchymal cells with long cytoplasmic processes, reaching a distance of tens to hundreds of microns from the cell body. Telocytes have recently emerged as an important intestinal stem cell niche component, providing Wnt proteins that are essential for stem and progenitor cell proliferation.
Although protocols on how to isolate mesenchyme from the mouse intestine are available, it is not clear whether these procedures allow the efficient isolation of telocytes. Isolating telocytes efficiently requires special protocol adjustments that would allow dissociation of the strong cell-cell contact between telocytes and neighboring cells without affecting their viability. Here, available intestinal mesenchyme isolation protocols were adjusted to support the successful isolation and culture of mesenchyme containing a relatively high yield of viable single-cell telocytes.
The obtained single-cell suspension can be analyzed by several techniques, such as immunostaining, cell sorting, imaging, and mRNA experiments. This protocol yields mesenchyme with sufficiently conserved antigenic and functional properties of telocytes, and can be used for several applications. For example, they can be used for co-culture with mouse- or human-derived organoids to support organoid growth with no growth factor supplementation, to better reflect the situation in the original tissue.

Here, we present a protocol to isolate murine intestinal mesenchyme, including telocytes. These can be used for several applications, such as co-culture with mouse or human-derived organoids, to support growth and better reflect the situation in the original tissue.

The murine small intestine, or colon mesenchyme, is highly heterogenous, containing distinct cell types including blood and lymphatic endothelium, nerves, ...

Assessing Strength Exercise Volume and Its Impact on Insulin Sensitivity in Obesity

Randomized Controlled Trial to Study the Acute Effects of Strength Exercise on Insulin Sensitivity in Obese Adults

An acute session of strength exercise (SE) ameliorates insulin sensitivity (IS) for several hours; however, the effects of SE volume (i.e., number of sets) have not been studied thoroughly. Although it is intuitive that some SE is better than none, and more is better than some for the improvement of IS, high-volume sessions might be challenging for diseased populations to complete, especially obese adults, for whom even a brisk walk can be challenging. This protocol details a randomized clinical trial to assess the acute effects of SE on IS in obese adults. The inclusion criteria are body mass index &#62;30 kg/m2, central obesity (waist circumference &#62;88 cm and &#62;102 cm for women and men, respectively), and age &#62;40 years. Participants will be familiarized with the SE (7 exercises targeting major muscle groups) and then will perform three sessions in a randomized order: session 1 - high-volume session (3 sets/exercise); session 2 - low-volume session (1 set/exercise); session 3 - control session (no exercise). Diet will be controlled the day before and on the day of the sessions. Sessions will be completed at night, and an oral glucose tolerance test will be performed the next morning, from which several indexes of IS will be derived, such as the area under the curve (AUC) of glucose and insulin, the Matsuda index, the Cederholm index, the muscle IS index, and the Gutt index. Based on pilot studies, we expect ~15% improvement in IS (insulin AUC, and Matsuda and Cederholm indexes) after the high-volume session, and ~8% improvement after the low-volume session compared to the control session. This study will benefit individuals who find high-volume SE sessions challenging but still aim to improve their IS by investing 1/3 of their time and effort.

Author Spotlight: Exploring the Impact of Reduced Resistance Exercise Volume on Metabolic Health 

This study describes a randomized controlled trial protocol aiming at assessing the acute effects of strength exercise volume on insulin sensitivity in obese individuals.

An acute session of strength exercise (SE) ameliorates insulin sensitivity (IS) for several hours; however, the effects of SE volume (i.e., number of ...

Establishment of 3D Morphogenesis in a Canine IBD Gut-on-a-Chip System

Three-Dimensional Morphogenesis in Canine Gut-on-a-Chip Using Intestinal Organoids Derived from Inflammatory Bowel Disease Patients

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal disorders similar to humans. Overcoming the inherent limitation of three-dimensional (3D) organoids in accessing the apical surface of the intestinal epithelium has led to the generation of two-dimensional (2D) monolayer cultures, which expose the accessible luminal surface using cells derived from the organoids. The integration of these organoids and organoid-derived monolayer cultures into a microfluidic Gut-on-a-Chip system has further evolved the technology, allowing for the development of more physiologically relevant dynamic in vitro intestinal models.
In this study, we present a protocol for generating 3D morphogenesis of canine intestinal epithelium using primary intestinal tissue samples obtained from dogs affected by inflammatory bowel disease (IBD). We also outline a protocol for generating and maintaining 2D monolayer cultures and intestine-on-a-chip systems using cells derived from the 3D intestinal organoids. The protocols presented in this study serve as a foundational framework for establishing a microfluidic Gut-on-a-Chip system specifically designed for canines. By laying the groundwork for this innovative approach, we aim to expand the application of these techniques in biomedical and translational research, aligning with the principles of the One Health Initiative. By utilizing this approach, we can develop more physiologically relevant dynamic in vitro models for studying intestinal physiology in both dogs and humans. This has significant implications for biomedical and pharmaceutical applications, as it can aid in the development of more effective treatments for intestinal diseases in both species.

Author Spotlight: Development and Application of a Canine IBD Gut-on-a-Chip Model for 3D Intestinal Morphogenesis Studies 

Integration of canine intestinal organoids and a Gut-on-a-Chip microfluidic system offers relevant translational models for human intestinal diseases. Protocols presented allow for 3D morphogenesis and dynamic in vitro modeling of the gut, aiding in the development of effective treatments for intestinal diseases in dogs and humans with One Health.

Canine intestines possess similarities in anatomy, microbiology, and physiology to those of humans, and dogs naturally develop spontaneous intestinal ...