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How do you determine the selectivity of your ROS dyes?

Posted September 18, 2020


For example, Cat#22903 uses your ROS Brite™ 670 sensor to quantify ROS in live cells. How do you determine its sensitivity to superoxide anions and hydroxyl radicals over other ROS and RNS such as peroxynitrite, organic hydroperoxyl, nitrogen dioxide, etc.?

Answer

We perform selectivity testing on all of ROS products. This is done by measuring the fluorescence intensity of each probe upon the subsequent addition of various ROS or RNS.

When testing Cat#22903’s ROS Brite™ 670 sensor, we introduced the sensor to a variety of ROS and RNS. When ROS Brite™ 670 reacted with hydroxyl radicals (•-OH) or superoxide anions (O-2) the fluorescence signal generated was very strong. In response to H2O2, TBHP, and –OCL the fluorescence signal was weak. And in response to NO or ONOO- there was no fluorescence signal.

Brief outline of how oxygen species were generated. For more detailed instruction refer to link in the Additional Resources located below:

  • ONOOwas synthesized from NaNO2 and H2O2
  • •-OH was synthesized by the Fenton reaction of H2O2and Fe2+
  • O-2 was synthesized from xanthine/xanthine oxidase system.
  • A stock solution of DEA/NONOate was prepared in 0.01 M NaOH solution to generate NO.
Additional resources

https://www.sciencedirect.com/science/article/pii/S0003269717300015

https://www.aatbio.com/products/cell-meter-fluorimetric-intracellular-total-ros-activity-assay-kit-deep-red-fluorescence?unit=22903

https://www.aatbio.com/products/ros-brite-670-optimized-for-detecting-reactive-oxygen-species-ros?unit=16002