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

Overview

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.

Protocol

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).

1. Preparation of NGM Plates for Testing the Effects of a Chemical (DIM) on the Intestinal Permeability of C. elegans Fed with P. aeruginosa
   1. 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).
   2. 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).
   3. Autoclave the two bottles (from steps 1.1–1.2), one empty 100 mL glass bottle, and one empty 500 mL glass bottle for 15 min at 121 °C. Then, allow the medium containing bottles to cool to 55 °C in the water bath for 30 min. Keep the agar medium in the water bath and remove the bottle containing broth (from step 1.2) for the next step.
   4. 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 °C.
   5. Label the autoclaved empty 500 mL glass bottle with dimethyl sulfoxide (DMSO) and the autoclaved empty 100 mL glass bottle with DIM.
   6. Transfer 50 mL of NGM broth into the DIM-labeled 100 mL bottle. Add 500 µL of 20 mM DIM stock into the bottle and mix well.
      NOTE: DIM is dissolved in DMSO (20 mM DIM stock).
   7. 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.
   8. 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 µM.
   9. 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.
   10. Add 150 mL of the NGM agar medium to the DMSO-labeled bottle and mix thoroughly.
   11. 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%.
   12. Solidify the plates at room temperature for at least 3 h and store them at 4 °C until use.
       NOTE: The protocol can be paused here.
   13. Remove the E. coli OP50 or P. aeruginosa PAO1 culture from the 4 °C refrigerator and vortex the culture properly before spreading onto the NGM plates.
   14. Put 800 µL 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 °C incubator overnight.
       NOTE: The protocol can be paused here. For the third experiment (Figure 1), 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.
2. Treatment of Bacteria or DIM and FITC-dextran Feeding
   1. Incubate the age-synchronized eggs at 20 °C for 64 h on NGM plates supplemented with live E. coli OP50 as food.
   2. 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. Incubate them at 20 °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).
   3. 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 µL 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 μg/mL.
   4. Prepare five E. coli OP50-containing NGM plates without FITC-dextran for the vehicle control treatment. For this purpose, divide 100 µL 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.
   5. After 48 h of treatment (from step 2.2), 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 (14–15 h).
      NOTE: For each treatment group, 5 replicates of FITC-dextran staining (or vehicle control feeding) are required.
   6. 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.
   7. Add 50 µL 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 µL 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.
3. 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.
   1. Capture fluorescence images and measure the fluorescence intensity using the Operetta High-Content Imaging System and analyze the images with Harmony software.
   2. In the Harmony software, press the icon Open lid to open the lid and to put the plate into the machine.
   3. Set up the parameters.
   4. Click Set up, select the plate type (96-well corning flat-bottom) and add the channels (bright-field and EGFP channels).
   5. Adjust the layout. Go to the Layout selection, and then select Track and adjust the parameters (first picture at 1 µm, number of planes are 10, distance is 1 µm).
   6. 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.
   7. 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.
   8. Go to Run experiment again and enter the plate name, then press Start to begin processing.
   9. 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 >200 µm², 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.
   10. Go to the Evaluation section to measure the intensity by obtaining a heatmap and data table. Set the Readout parameter to Cells — Intensity Cell EGFP Mean — Mean per Well and start the evaluation.
       NOTE: The protocol can be paused here.
   11. To extract the data from Operetta, click the Setting button and choose Data management.
   12. Choose Write archive, and then open the browse and select the file.
   13. To select the file, click the small + signal at the left corner, and then click Measurement and choose Plate name.
   14. Select the file and click OK.
   15. Select the path to save the file by clicking the Browse signal at the active path.
   16. Click Start to save the data file.
       NOTE: The protocol can be paused here.
4. Statistical Analysis of the FITC-dextran Fluorescence of C. elegans
   1. Import the data and calculate the mean and standard deviation (SD) using a statistics software.
   2. Analyze the significant difference with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test.

Representative Results

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.

Materials

Name	Company	Catalog Number	Comments
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