This study investigates the impact of bacterial colonization on the oxidative stress response in C. elegans. By utilizing fluorescence assays, the research assesses how different bacteria affect the gut epithelium and the subsequent release of amino acid derivatives.
Begin with a tube containing C. elegans worms.
Transfer the worms to a multiwell plate with nutrient-rich agarose media containing either the control bacteria or the test bacteria.
During incubation, the worms ingest bacteria, which colonize the gut epithelium and release amino acids that serve as a nutrient source. These amino acids are metabolized into amino acid derivatives and stored in lysosome-related gut granules.
Next, add fresh buffer and transfer the worms to a black multiwell assay plate.
Add a chemical stress inducer to each well and seal the plate.
Place the plate in a fluorescence plate reader and record the fluorescence.
In worms, the stress inducer causes oxidative stress, which damages the gut epithelial cells.
This causes the lysosome-related gut granules to rupture, releasing amino acid derivatives, which, upon exposure to ultraviolet light, fluoresce blue.
Compare the fluorescence of worms exposed to the control and test bacteria to assess bacterial colonization efficiency and their impact on the host's oxidative stress response.
Adjust worm concentration to 15 worms per microliter in the 15 milliliter conical tube. Then, transfer 8 microliters of worm solution into each of the wells of the eight 96-well NGM agarose plates using a multi-channel pipette or repeat pipette.
After 36 hours, dispense 30 microliters of M9 into each well of the 96-well plate. Next, transfer worms to the 384-well plate according to set layouts using low retention tips, ensuring that the plate readers are set up properly. Afterward, top up the 384-well plates with more M9, aiming for a final volume of 60 microliters per well by adding 40 microliters of M9 for thermal stress assay and 34 microliters of M9 in 6 microliters of TBHP for TBHP-induced oxidative stress.
Then, start the assay within two minutes of adding TBHP. Next, close the plates with their transparent lid, and seal the edges of the 384-well plates with masking tape, ensuring that the tape does not go over the lid or under the plate. Then, insert the plate into the plate reader.
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