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.col