This method will be helpful for basic biological studies related to the interaction between intestinal microbiota and gut health. In addition, this protocol may be used for screening potential probiotics and nutraceuticals to cure intestinal health problems, such as leaky gut syndrome and inflammatory bowel diseases. This protocol uses a high-throughput imaging system to enable rapid and precise quantitative analysis of intestinal permeability in C.elegans treated with various bacteria and chemicals.
We recommend that a scientist who is not familiar with this technique limits the number of samples in order to avoid mislabeling and other mistakes. This procedure consists of many steps that require visual demonstration, such as transferring worms to 96-well plates and using the Operetta machine, as well as the Harmony software. Start by preparing 500 milliliters of sterile LB medium.
Inoculate a single colony of P.aeruginosa into the medium, and incubate the culture for 14 to 15 hours at 37 degrees Celsius while shaking at 150 rpm. The next day, evenly distribute the bacterial culture into two 500-milliliter tubes, and centrifuge them at 3, 220 times g and four degrees Celsius for 30 minutes. Remove the supernatant, leaving 25 milliliters in each tube, for a total remaining volume of 50 milliliters, then resuspend the bacterial pellet.
Store the bacterial culture at four degrees Celsius until ready to use. To test the effects of bacteria on the intestinal permeability of C.elegans, remove the cultures from the refrigerator, and thoroughly vortex them. Add a total of 800 microliters to each fresh NGM plate, and allow the plates to dry in a 20-degree Celsius incubator overnight.
To prepare DIM plates, add 50 milliliters of NGM agar medium into a bottle of DIM containing NGM broth, and mix thoroughly. Then, pour 20 milliliters of the medium into each 90-by-15-millimeter Petri dish. Prepare DMSO plates by adding 20 milliliters of DMSO containing NGM medium to each Petri dish, and leave the plates at room temperature for at least three hours to solidify.
Spread 800 microliters of vortexed E.coli or P.aeruginosa culture onto each fresh NGM plate, and allow the plates to dry in a 20-degree Celsius incubator overnight. Wash the age-synchronized L4 larvae with S-buffer, and transfer approximately 500 worms to the NGM plates containing different bacteria and chemicals. Then, incubate the plates at 20 degrees Celsius for 48 hours.
Prepare the FITC-dextran-supplemented plates by mixing two milliliters of heat-inactivated E.coli with four milligrams of FITC-dextran. Add 100 microliters of the mixture to each of 20 fresh NGM agar plates, and allow the plates to dry for one hour on a clean lab bench. After 48 hours of treatment, wash the worms with S-buffer, transfer them to FITC-dextran-supplemented plates and to NGM plates without FITC-dextran, and incubate the plates overnight.
The next day, wash the worms with S-buffer, and allow them to crawl in the fresh NGM agar plate for one hour. Add 50 microliters of 4%formaldehyde to each well of a black, 96-well, flat-bottomed plate, and transfer approximately 50 worms into each well. After one to two minutes, remove all the formaldehyde, and add 100 microliters of mounting medium into each well.
Formaldehyde is a toxic chemical and should be handled with care. To capture fluorescent images, press the Open Lid icon within the imaging software, and insert the plate into the machine. Click Setup, select the plate type, and add the brightfield and EGFP channels.
To adjust the layout, go to the Layout selection, and select Track. Set the first picture at one micrometer, number of planes to 10, and the distance to one micrometer. Select one of the treatment wells and one capture field, and press Test in the Run Experiment section.
If the images are satisfactory, return to the Setup section, and press the Reset icon. Then, select all the target wells and a suitable number of capture fields. Go to Run Experiment, enter the plate name, and press Start.
To measure fluorescence intensity, go to the Image Analysis section, input the image, and find the cell by choosing the EGFP channel and method B.Adjust the parameters according to the manuscript directions, and choose the mean as the output. Press the Apply icon to save the setup. Obtain a heat map and a data table by going to the Evaluation section and adjust the readout parameter, then start the evaluation.
C.elegans showed a significant increase in FITC-dextran fluorescence after incubation with P.aeruginosa compared with the other two bacterial strains, indicating that P.aeruginosa caused more vital damage to the epithelial gut barrier and increased intestinal permeability. Live and heat-inactivated P.aeruginosa triggered very different effects on the intestinal permeability in C.elegans. Upon heat inactivation, both the fluorescence images and statistical data indicate that P.aeruginosa did not trigger any toxicity to nematodes.
A 48-hour co-treatment with DIM significantly decreased the FITC-dextran fluorescence intensity inside the guts of worms exposed to P.aeruginosa, which demonstrates that DIM can be considered a good natural product to cure intestinal permeability dysfunction caused by bacterial infections. It's important to remember that during the preparation of the FITC-dextran-coated plates, you need to minimize the light exposure because of the light sensitivity of FITC-dextran. This technique can be used to determine the pathogenic and probiotic effects of intestinal bacteria and their active components by testing the bacteria, as well as their chemical composition.
