Hi Hsuan zi Chung ,

This is just a thought. Let me know if this makes sense.

1. Prepare plastic particles compounded with BP-8: Create plastic particles compounded with BP-8 by incorporating the photoinitiator into the plastic matrix during particle synthesis or coating the particles with BP-8. Optimize the concentration of BP-8 to ensure an appropriate ROS-generating effect without excessive toxicity.

2. Obtain zebrafish embryos: Obtain zebrafish embryos at the desired developmental stage (e.g., early embryogenesis) for exposure experiments. Follow established protocols for zebrafish husbandry and embryo collection.

3. Exposure setup: Create an exposure system where zebrafish embryos are exposed to the plastic particles compounded with BP-8. This can involve placing the embryos in a solution containing the particles or directly exposing them to a controlled concentration of the particles.

4. ROS detection: There are several methods to measure ROS levels in zebrafish embryos exposed to the plastic particles. Some common techniques include:

a. Dihydroethidium (DHE) staining: DHE is a fluorescent dye that becomes oxidized and emits red fluorescence in the presence of ROS. Incubate the exposed zebrafish embryos with DHE, followed by imaging or fluorescence detection to quantify ROS levels.

b. Dichlorofluorescein diacetate (DCF-DA) assay: DCF-DA is a fluorescent dye that is deacetylated by cellular esterases and reacts with ROS to produce fluorescent DCF. Incubate the exposed zebrafish embryos with DCF-DA, followed by imaging or fluorescence detection to measure ROS levels.

c. Electron paramagnetic resonance (EPR) spectroscopy: EPR spectroscopy directly measures the unpaired electrons in ROS. It requires specialized equipment and expertise. With this method, the exposed zebrafish embryos are homogenized, and the resulting homogenate is analyzed using EPR spectroscopy to quantify ROS levels.

d. Other assays: There are additional ROS-specific assays, such as Amplex® Red or hydroxyphenyl fluorescein (HPF) assays, that can be adapted to measure ROS in zebrafish embryos. These assays use fluorescent or colorimetric probes that react specifically with ROS and can be detected using fluorescence or absorbance measurements.

5. Data analysis: Analyze the collected data to quantify ROS levels in the zebrafish embryos exposed to the plastic particles. Use appropriate controls, statistical analysis, and comparison to determine significant differences in ROS production.

Use roGFP expressing transgenic zebrafish. You can then quantify ROS directly.

How to stain if using DCF-DA staining method?

How long does it take to dye?