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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.
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.
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 stai....
1. Preparation of P. aeruginosa PAO1 and Escherichia coli OP50 Culture
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 ± 6.6, 369.7 ± 38.9, and 105.6 ± 10.6%, respectively. The data emphasize that P. aeru.......
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.......
This study was supported by a Korea Institute of Science and Technology intramural research grant (2E29563).
....Name | Company | Catalog Number | Comments |
3,3’-diindolylmethane | Sigma | D9568 | |
90×15 mm Petri dishes | SPL Life Sciences, South Korea | 10090 | |
60×15 mm Petri dishes | SPL Life Sciences, South Korea | 10060 | |
Bactor Agar | Beckton Dickinson | REF. 214010 | |
Formaldehyde solution | Sigma | F1635 | |
Brain Heart Infusion (BHI)Â | Becton Dickinson | REF. 237500 | |
Caenorhabditis elegans N2 | Caenorhabditis Genetics Center (CGC) | Wild type | |
Cholesterol | Sigma | C3045 | |
Costa Assay Plate, 96 Well Black With Clear Flat Bottom Non-treated, No Lid Polystyrene | Corning Incorporated | REF. 3631 | |
Dimethyl sulfoxide | Sigma | D2650 | |
Enterococcus faecalis KCTC 3206 | Korean Collection for Type Culture | KCTC NO. 3206 | Falcutative anaerobic |
Escherichia coli OP50 | Caenorhabditis Genetics Center (CGC) | ||
Fluorescein isothiocyanate - dextran | Sigma | FD10S | |
Harmony software | PerkinElmer | verson 3.5 | |
Luria-Bertani LB medium | Merck | VM743185 626Â 1.10285.5000 | |
Magnesium sulfate heptahydrate | Fisher Bioreagents | BP2213-1 | |
Fluoromount aqueous mounting medium | Sigma | F4680 | |
Operetta CLS High-Content Analysis System | PerkinElmer | Â HH16000000 | |
Peptone | Merck | EMD 1.07213.1000 | |
Pseudomonas aeruginosa PA01 | Korean Collection for Type Culture | KCTC NO. 1637 | |
Sodium Chloride | Fisher Bioreagents | BP358-1 | |
Stereo Microscope | Nikon, Japan | SMZ800N | |
Yeast extract | Becton Dickinson | REF. 212750 |
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