One of my colleagues had a nice neutrophil response assay working about 3 years ago after copper sulfate hair cell damage. Recently we have not been able to recapitulate these previous results. We are using the same lyzc:dsred and lyzc:gfp lines as before but see little to no response after copper sulfate treatment. Any insight would be helpful.
The damage to hair cells induced by copper sulfate occurs rapidly, as copper ions quickly enter through the MET (mechanotransduction) channels of the hair cells. Therefore, observations should be conducted within 40–50 minutes after exposure to 10 -20μM CuSO₄. The typical developmental stage used is 5 dpf, and live imaging is performed under tricaine anesthesia.
This process occurs in distinct stages: first, the damage to hair cells; then, the migration of neutrophils to the injury site. You may start by observing whether hair cell damage occurs. A common approach is to perform DASPEI staining for 15 minutes, followed by live imaging under a fluorescence microscope.
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