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><tbody><tr><td>3,3&amp;rsquo;-diindolylmethane&amp;nbsp;</td><td>Sigma</td><td>D9568</td><td></td></tr><tr><td>90&amp;times;15 mm Petri dishes</td><td>SPL Life Sciences, South Korea</td><td>10090</td><td></td></tr><tr><td>60&amp;times;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&amp;nbsp;</td><td>Sigma</td><td>F1635</td><td></td></tr><tr><td>Brain Heart Infusion (BHI)&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp;</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&amp;nbsp; 1.10285.5000</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate&amp;nbsp;</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>&amp;nbsp;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...

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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 ...

measuring-effects-bacteria-chemicals-on-intestinal-permeability

Research

JoVE Journal

Medicine

14.2K Views.  Korea Institute of Science and Technology. Deze methode zal nuttig zijn voor fundamentele biologische studies met betrekking tot de interactie tussen darmmicrobiota en darmgezondheid. Bovendien, Dit protocol kan worden gebruikt voor screening potentiële probiotica en nutraceuticals om darmgezondheidsproblemen te genezen, zoals lekkende darm syndroom en inflammatoire darmziekten. Dit protocol maakt gebruik van een beeldvormingssysteem met hoge doorvoer om een snelle en nauwkeurige kwantitatieve analyse van de darmdoorlaatbaarheid in C...

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

Measuring the Strength of Mice

Kondziela7 devised the inverted screen test and published it in 1964. It is a test of muscle strength using all four limbs. Most normal mice easily score maximum on this task; it is a quick but insensitive gross screen, and the weights test described in this article will provide a finer measure of muscular strength.
There are also several strain gauge-based pieces of apparatus available commercially that will provide more graded data than the inverted screen test, but their cost may put them beyond the reach of many laboratories which do not specialize in strength testing. Hence in 2000 a cheap and simple apparatus was devised by the author. It consists of a series of chain links of increasing length, attached to a "fur collector" a ball of fine wire mesh sold for preventing limescale build up in hard water areas. An accidental observation revealed that mice could grip these very tightly, so they proved ideal as a grip point for a weight-lifting apparatus. A common fault with commercial strength meters is that the bar or other grip feature is not thin enough for mice to exert a maximum grip. As a general rule, the thinner the wire or bar, the better a mouse can grip with its small claws.
This is a pure test of strength, although as for any test motivational factors could potentially play a role. The use of scale collectors, however, seems to minimize motivational problems as the motivation appears to be very high for most normal young adult mice.

Deficits in muscular strength occur in many clinical conditions such as motor neuron disease. The inverted screen and weight lifting tests described here measure strength in mice almost exclusively, with minimal influence of factors such as coordination.

Kondziela7 devised the inverted screen test and published it in 1964. It is a test of muscle strength using all four limbs. Most normal mice easily score ...

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term effects are very important in judging their consequences in the environment and on human health. In demonstrating long-term influences, effects of chemicals over generations in recent studies provide new insight. Here, we describe protocols for studying effects of chemicals over multiple generations using free-living nematode Caenorhabditis elegans. Two aspects are presented: (1) trans-generational (TG) and (2) multi-generational effect studies, the latter of which is separated to multi-generational exposure (MGE) and multi-generational residual (MGR) effect studies. The TG effect study is robust with a simple purpose to determine whether chemical exposure to parents can result in any residual consequences on offspring. After the effects are measured on parents, sodium hypochlorite solutions are used to kill the parents and keep the offspring so as to facilitate effect measurement on the offspring. The TG effect study is used to determine whether the offspring are affected when their parent is exposed to the pollutants. The MGE and MGR effect study is systematical used to determine whether continuous generational exposure can result in adaptive responses in offspring over generations. Careful pick-up and transfer are used to distinguish generations to facilitate effect measurement on each generation. We also combined protocols to measure locomotion behavior, reproduction, lifespan, biochemical and gene expression changes. Some example experiments are also presented to illustrate the trans- and multi-generational effect studies.

Using Caenorhabditis elegans for Studying Trans- and Multi-Generational Effects of Toxicants

Trans- and multi-generational effects of persistent chemicals are essential in judging their long-term consequences in the environment and on the human health. We provide novel detailed methods for studying trans- and multi-generational effects using free-living nematode Caenorhabditis elegans.

Information about toxicities of chemicals are essential in their application and waste management. For chemicals at low concentrations, the long-term ...

Environment

High-Throughput Assay to Measure Caenorhabditis elegans Food Intake and Lifespan

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of Caenorhabditis elegans&#160;(C. elegans), an important parameter when studying the genetics of aging or metabolism. In most species, feeding is determined by measuring the difference between the amount of food provided and the amount left after a given time interval. The method presented here uses the same strategy to determine the feeding of C. elegans. It measures the amount of bacteria, the food source of C. elegans, cleared within 72 h. This method uses 96-well microtiter plates and has allowed the screening of hundreds of drugs for their ability to modulate food intake at a speed and depth not possible in other animal models. The strength of this assay is that it allows to measure feeding and lifespan simultaneously and directly measures the disappearance of food and, thus, is based on the same principles used for other organisms, facilitating species-to-species comparison.

Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF 

This protocol describes an assay to quantify Caenorhabditis elegans feeding rate based on measuring the clearance of bacteria in liquid culture.

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

Behavior

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 ...

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

C. elegans egg-laying behavior is affected by environmental cues such as osmolarity1 and vibration2. In the total absence of food C. elegans also cease egg-laying and retain fertilized eggs in their uterus3. However, the effect of different sources of food, especially pathogenic bacteria and particularly Enterococcus faecalis, on egg-laying
	behavior is not well characterized. The egg-in-worm (EIW) assay is a useful tool to quantify the effects of different types of bacteria, in this case E. faecalis, on egg- laying behavior.
	
	EIW assays involve counting the number of eggs retained in the uterus of C. elegans4. The EIW assay involves bleaching staged, gravid adult C. elegans to remove the cuticle and separate the retained eggs from the animal. Prior to bleaching, worms are exposed to bacteria (or any type of environmental cue) for a fixed period of time. After bleaching, one is very easily able to count the number of eggs retained inside the uterus of the worms. In this assay, a quantifiable increase in egg retention after E. faecalis exposure can be easily measured. The EIW assay is a behavioral assay that may be used to screen for potentially pathogenic bacteria or the presence of environmental toxins. In addition, the EIW assay may be a tool to screen for drugs that affect neurotransmitter signaling since egg-laying behavior is modulated by neurotransmitters such as serotonin and acetylcholine5-9.

Measuring the Effects of Bacteria on C. Elegans Behavior Using an Egg Retention Assay

An egg-in-worm (EIW) assay is a useful method to quantify egg-laying behavior. Alterations in egg laying can be a behavioral response of the model organism Caenorhabditis elegans to potentially harmful environmental substances such as those produced by pathogenic bacteria.

C. elegans egg-laying behavior is  affected by environmental cues such as osmolarity1 and  vibration2. In the total absence of food C. elegans also cease ...

Biology

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome

The composition of the gut microbiome can have a dramatic impact on host physiology throughout the development and the life of the animal. Measuring compositional changes in the microbiome is crucial in identifying the functional relationships between these physiological changes. Caenorhabditis elegans has emerged as a powerful host system to examine the molecular drivers of host-microbiome interactions. With its transparent body plan and fluorescent-tagged natural microbes, the relative levels of microbes within the gut microbiome of an individual C. elegans animal can be easily quantified using a large particle sorter. Here we describe the procedures for the experimental setup of a microbiome, collection, and analysis of C. elegans populations in the desired life stage, operation, and maintenance of the sorter, and statistical analyses of the resulting datasets. We also discuss considerations for optimizing sorter settings based on the microbes of interest, the development of effective gating strategies for C. elegans life stages, and how to utilize sorter capabilities to enrich animal populations based on gut microbiome composition. Examples of potential applications will be presented as part of the protocol, including the potential for scalability to high-throughput applications.

Application of Flow Vermimetry for Quantification and Analysis of the Caenorhabditis elegans Gut Microbiome

Caenorhabditis elegans is a powerful model to examine the molecular determinants driving host-microbiome interactions. We present a high throughput pipeline profiling the single animal levels of gut microbiome colonization together with key aspects of the C. elegans physiology.

The composition of the gut microbiome can have a dramatic impact on host physiology throughout the development and the life of the animal. Measuring ...

Evaluating Metabolism and Aging Pattern in Caenorhabditis elegans

Using Low-Cost Dyes to Visualize Glycogen Accumulation and Gut Integrity in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences teaching through the staining of the cells or their molecular content. Lugol dye (iodine-potassium iodide solution) has been widely used in biochemistry to stain glycogen stores. In this context, it is possible to observe differences between fed and starved animals, besides the effects of different conditions, such as different diets and oxygen levels. Erioglaucine is a blue dye that indicates the loss of the intestinal barrier. When the intestinal barrier is intact, the blue dye stains inside the lumen; however, when this integrity is disrupted, the dye leaks into the body cavity. Using a stereomicroscope or a microscope, teachers can demonstrate physiological and biochemical alterations, or they can instigate students to ask a scientific question and hypothesize and test their hypothesis using these assays. The present protocol describes two staining techniques in C. elegans that can be easily carried out by students.

Author Spotlight: Exploring Metabolic and Aging Processes in C. elegans Using Low-Cost, High-Impact Assays

Teaching biological sciences can be made more stimulating for students through the use of experimentation. This manuscript presents two different yet complementary protocols that can be utilized in the classroom to encourage students to formulate and test hypotheses related to high-calorie diets, starvation, and aging.

Caenorhabditis elegans (C. elegans) is a transparent, non-parasitic nematode with a simple biology, which makes it a great tool for biological sciences ...

FITC-Dextran Feeding: A Method to Quantify Intestinal Permeability in C. elegans

Source: Le, T. A. N., et al. <a href="https://www.jove.com/video/60419/measuring-effects-bacteria-chemicals-on-intestinal-permeability">Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of&#160;Caenorhabditis elegans.</a>&#160;J. Vis. Exp. (2019).
This video introduces a method of to visualize and measure the integrity of the C. elegans intestine lumen by feeding worms FITC-labeled dextran.&#160; The example protocol shows the assay done with 3,3&#8217;-diindolylmethane (DIM)-treated and pathogen-fed worms.

Source: Le, T. A. N., et al. Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of&#160;Caenorhabditis elegans.&#160;J. Vis. ...

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

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