Visualizing Pathogen-Induced Oxidative Stress in C. elegans Using GFP-Tagged SKN-1

Begin with two plates containing C. elegans worms that express green fluorescent protein-tagged SKN-1, a transcription factor localized in the intestinal cytoplasm. These worms also contain intestinal lipofuscin granules that show autofluorescence.

One plate is seeded with non-pathogenic bacteria as the control group, the other with pathogenic bacteria as the test group.

In the test group, hydrogen peroxide produced by the pathogen induces oxidative stress, triggering SKN-1 translocation to intestinal nuclei.

In the control group, the majority of SKN-1 remains diffusely distributed in the cytoplasm.

Wash each plate with a buffer and transfer the worms into tubes.

Centrifuge to separate the worms and remove the buffer.

Add a medium containing sodium azide to anesthetize them.

Mount the worms on agarose pads, place coverslips, and observe under a fluorescence microscope. Use autofluorescence to visualize the nuclear boundaries.

Strong nuclear SKN-1 signal in test, relative to the diffuse cytoplasmic signal in controls, indicates nuclear relocalization and confirms pathogen-induced oxidative stress.

Using a pipette, add approximately 100 L4 larvae to each THY streptococcus seeded and NGM E.coli seeded plates. Use three plates per strain of bacteria.

Incubate the plates for 2 to 3 hours at 25 degrees Celsius. Thereafter, remove the plates from the incubator. Wash them with M9W and collect the worms in 15 milliliter conical tubes.

Wash the worms three times as done previously in the centrifugal step. Remove most of the M9W by aspiration. And add 500 microliters of M9W containing 2 millimolar sodium azide or tetramisole hydrochloride to the worm pellet for anesthesia.

Incubate the tube with worm pellets at room temperature for 15 minutes. Then, drop 15 microliters of the worm suspension on to a prepared agarose pad. Under a fluorescent microscope, visualize the localisation of SKN-1 utilizing FITC and DAPI filters.

Image worms at 10 times and 20 times magnifications. Score the worms based on three levels of localisation; low localisation, where no nuclear localisation is observed, medium localisation, with SKN-1 BCGFP in the anterior or posterior of the worm, and high localisation, where nuclear localisation of SKN-1 BCGFP is observed in all intestinal cells.