Does anyone have any in-vivo or ex-vivo protocols for ROS measurement in C. elegans?

02 October 2023 1 5K Report

I have some RNAi that I believe affect ROS production and would like to test this. I rear N2 on this RNAi and let worms grow until L4. I then treat these worms at L4 in 96-well plate (~100 worms/well) with Rotenone and Juglone for ~8h. Then, I add 50uM DCFDA or 5uM DHE to the well and stain for 1h. I then image worms in Cytation. However, this has not worked. Does anybody have any suggestions?

Robert Adolf Brinzer

You can use the Amplex Red kit and follow the instructions or use a roGFP/mito-roGFP expressing line and then quantify using whole worm homogenate and a plate reader or fluorescence microscopy if your setup supports measuring two emission frequencies.

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