Why does null hypothesis testing matter for FITC-dextran permeability assays?

Null hypothesis testing enables objective determination of whether observed differences in intestinal fluorescence reflect true permeability changes or random variation, supporting rigorous target validation.

How does independent variable isolation fit the FITC-dextran workflow?

Isolating variables such as bacterial strain or compound exposure ensures that changes in fluorescence are attributable to the intervention, strengthening mechanistic insights in the discovery pipeline.

What do quantitative dependent variable measurements enable in this assay?

Quantitative fluorescence measurements allow precise comparison of intestinal permeability across experimental groups, enabling robust statistical analysis and data-driven decision making.

Why are replication requirements critical for cross-functional collaboration in permeability studies?

Replication ensures that permeability findings are reproducible and reliable, facilitating data sharing and alignment across discovery, screening, and translational teams.

What statistical analysis capabilities are required before implementing FITC-dextran readouts?

Teams must be equipped to perform statistical comparisons of fluorescence intensity, account for background autofluorescence, and validate significance thresholds to support actionable R&D decisions.

FITC-Dextran Feeding: een methode om intestinale permeabiliteit te kwantificeren bij C. elegans

JoVE Encyclopedia of Experiments

Biologie

0 weergaven • 2:56 min • April 30th, 2023

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

- C. elegans is used as an experimental model of infection by intestinal pathogens which cause disease, in part, by disrupting the intestinal barrier, increasing its permeability. To quantify barrier integrity after exposure to 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 to eat the labeled bacteria. Those with intact intestinal barriers will rapidly clear the food, while in worms with more permeable intestines, the fluorescent marker will penetrate beyond the lumen of the organ.

Transfer worms to a formaldehyde solution to fix the animals. After 1 to 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 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.

Gerelateerde video's

0 Bekeken

Laatst bijgewerkt: 1 augustus 2026

FITC-Dextran Feeding: een methode om intestinale permeabiliteit te kwantificeren bij C. elegans

Overzicht

Belangrijkste studiecomponenten

Wetenschapsgebied

Achtergrond

Doel van het onderzoek

Gebruikte methoden

Belangrijkste resultaten

Conclusies

Veelgestelde vragen

Executive Industry Relevance

Strategic Applications in Biopharma R&D

Early Discovery & Target Validation

Screening & Assay Development

Translational & Preclinical Research

Pipeline & Workflow Integration

Operational & Enterprise Impact

Implementation Considerations

Biopharma FAQs

C. elegans darm als een Model voor intercellulaire Lumen genuitdrukking en In Vivo gepolariseerde biogenese van eencellige niveau: Labeling door antilichaam kleuring, RNAi verlies-van-functie analyse en beeldvorming

Real-time meting van epitheliale barrière permeabiliteit in menselijke intestinale Organoids

In Vitro en In Vivo benaderingen te bepalen van intestinale epitheliale cel permeabiliteit

Functionele Evaluatie van darmmotiliteit en de darmwand ontsteking in Knaagdieren: Analyses in een gestandaardiseerd model van Intestinal Manipulatie

Assessment of Intestinal Permeability in C. elegans Following Bacterial Exposure

Ex Vivo Intestinale Sacs mucosale permeabiliteit beoordelen Modellen van gastro-intestinale aandoeningen

Het analyseren van gunstige effecten van voedingssupplementen op darmepitheel Barrier Functies tijdens de Experimental Colitis

Meten van de effecten van bacteriën en chemicaliën op de intestinale permeabiliteit van Caenorhabditis elegans

Een intravitale microscopie-gebaseerde benadering om darmdoorlatendheid en epitheelcelvergieten prestaties te beoordelen

Functionele beoordeling van intestinale permeabiliteit en neutrofiele transepitheliale migratie bij muizen met behulp van een gestandaardiseerd intestinale lusmodel