FITC-Dextran Feeding: A Method to Quantify Intestinal Permeability in C. elegans
Trascrizione
– C. elegans is used as an experimental model of infection by intestinal pathogens which cause disease, di part, by disrupting the intestinal barrier, increasing its permeability. To quantify barrier integrity after exposure a a bacteria or chemical compound of interest, collect worms by washing them off their plates.
Next, place these worms on agar plates with labeled food composed of heat-killed E. coli mixed with FITC-dextran, a high molecular weight polysaccharide with a fluorescent tag.
Allow the worms a eat the labeled bacteria. Those with intact intestinal barriers will rapidly clear the food, while di worms with more permeable intestines, the fluorescent marker will penetrate beyond the lumen of the organ.
Transfer worms a a formaldehyde solution a fix the animals. After 1 a 2 minutes, replace the formaldehyde with mounting media.
Finally, image the worms by fluorescent microscopy. Note that C. elegans intestinal granules are autofluorescent, so there will be background signal. But overall, more intestinal fluorescence indicates higher permeability.
In the example protocol, we will assay the effect of treatment with the biomolecule DIM on the intestinal permeability of worms infected with pathogenic Pseudomonas aeruginosa bacteria.
– Prepare the FITC-dextran-supplemented plates by mixing 2 milliliters of heat-inactivated E. coli with 4 milligrams of FITC-dextran. Add 100 microliters of the mixture a each of 20 fresh NGM agar plates and allow the plates a dry for one hour on a clean lab bench. After 48 hours of treatment, wash the worms with S Buffer. Transfer them a FITC-dextran supplemented plates and a NGM plates without FITC-dextran and incubate the plates overnight.
The next day, wash the worms with S Buffer and allow them a crawl di the fresh NGM agar plate for one hour. Add 50 microliters of 4% formaldehyde a each well of a black 96-well flat-bottomed plate and transfer approximately 50 worms into each well. After 1 a 2 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.
