Mode of Action

The assay to establish which commonly known molecular pathways are activated by test compounds in C. elegans.

Method description

For this assay we take advantage of the existing C. elegans GFP-reporter strains for a variety of key molecular stress and longevity pathways, as well as the capacity of our device to perform fluorescent imaging.

A synchronized population of C. elegans is injected into the microfluidic platform at the first larval stage (L1). Worms are then confined within dedicated microfluidic chambers and are continuously fed with an E. coli solution.

Worms are chronically exposed to the test compounds starting from the last larval stage prior to sexual maturity (L4) for 85 hours (day 3 of adulthood). The compounds to be tested are mixed with the E. coli solution. Freeze-dried OP50 E. coli are used as a food source for the whole duration of the experiment, preventing the metabolization of the tested molecules by the bacteria.

The images of each microfluidic chamber are recorded every hour by a fluorescence microscope. Time-resolved changes in intensities of the fluorescent signal are then extracted from the collected images.

Readouts

  • Autophagy
  • Oxidative stress
  • Endoplasmic reticulum Unfolded Protein Response
  • Mitochondrial Unfolded Protein Response
  • Heat shock response
  • Mitochondrial content in the muscle
  • Mitochondrial content in the intestine

Dynamics of mitochondrial unfolded protein response (UPRmt) activation in hsp-6::gfp C. elegans treated with the antibiotic doxycycline starting from the L1 larval stage (right) vs untreated worms (left), as observed via fluorescence imaging by the SydLab system.

Pathway activation (dynamics)

Temporal evolution of the average fluorescence intensity measured for hsp-6::gfp C. elegans treated with the antibiotic doxycycline starting from the L1 larval stage vs untreated worms (vehicle). Increased expression of the hsp-6::gfp reporter reveals the activation of the mitochondrial unfolded protein response (UPRmt) induced by the drug treatment.

Pathway activation (peak intensity)

Average peak fluorescence intensity for hsp-6::gfp C. elegans treated with the antibiotic doxycycline at different doses (data normalized to the value measured for the untreated worm population). Doxycycline treatments dose-dependently activate the mitochondrial unfolded protein response (UPRmt).

Assay combinations

The mode of action assay can be offered on its own or in combination with the following assays upon request.

Resources

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