Source: Le, T. A. N., et al. Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans. 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. The example protocol shows the assay done with 3,3’-diindolylmethane (DIM)-treated and pathogen-fed worms.
This protocol is an excerpt from Le et al, Measuring the Effects of Bacteria and Chemicals on the Intestinal Permeability of Caenorhabditis elegans, J. Vis. Exp. (2019).
Figure 1: The effect of DIM on the intestinal permeability of C. elegans fed P. aeruginosa. Microscopy images of worms from (A) E. coli OP50 without FITC-dextran feeding, (B) E. coli with FITC-dextran feeding, (C) P. aeruginosa PAO1 with FITC-dextran feeding, and (D) P. aeruginosa and DIM (100 µM) cotreatment with FITC-dextran feeding. Scale bar = 1 mm (white), and 200 µm (black). Age-synchronized L4 larvae were incubated for 48 h in NGM plates seeded with live E. coli (A, B), live P. aeruginosa (C), live P. aeruginosa and DIM (D). Then, the worms were transferred to plates containing FITC-dextran (B–D), except for the vehicle control (A). (E) The FITC fluorescence intensity indicates that the gut permeability of C. elegans was affected by DIM. Columns and error bars indicate the mean ± SD. ***P < 0.001 for significant difference from the vehicle control.###P < 0.001 and ##P < 0.01 for significant difference from the FITC-dextran-treated worms fed with P. aeruginosa PAO1 (ANOVA, n = 5). This graph is representative of two independent experiments.
3,3’-diindolylmethane | Sigma | D9568 | |
90×15 mm Petri dishes | SPL Life Sciences, South Korea | 10090 | |
Bactor Agar | Beckton Dickinson | REF. 214010 | |
Formaldehyde solution | Sigma | F1635 | |
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 | |
Escherichia coli OP50 | Caenorhabditis Genetics Center (CGC) | ||
Fluorescein isothiocyanate – dextran | Sigma | FD10S | |
Harmony software | PerkinElmer | verson 3.5 | |
Magnesium sulfate heptahydrate | Fisher Bioreagents | BP2213-1 | |
Fluoromount aqueous mounting medium | Sigma | F4680 | |
Operetta CLS High-Content Analysis System | PerkinElmer | 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 |