ROS detection was performed using the ROS assay-highly sensitive DCFH DA kit (Dojindo, Japan) following the manufacturer's protocol. Briefly, after washing the preformed on-chip microvasculature twice with Hank's balanced salt solution (HBSS; Gibco), the working solution of the ROS assay was introduced into channels two and four (day 0) and incubated for 30 min. The working solution was then removed, the microvasculature was washed twice with HBSS, the medium containing SS (3 μM) was added, and the chip was incubated for 1 h. Subsequently, the medium was removed, and the microvasculature was washed again with HBSS and imaged to observe fluorescence. Red fluorescent protein-labeled HUVECs were used in this experiment (Fig. S2).
Dcfh da kit
Manufactured by Dojindo Laboratories
Sourced in Japan
The DCFH-DA kit is a fluorescence-based reagent used for the detection and quantification of intracellular reactive oxygen species (ROS) in cells. It provides a simple and reliable method to measure cellular oxidative stress levels.
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2 protocols using dcfh da kit
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ROS Detection in Microvasculature
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Intracellular ROS Measurement Assay
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Intracellular ROS generation was determined with a ROS indicator dye dichlorodihydrofluorescein diacetate (DCFH-DA) kit (R252; Dojindo Laboratories). Acutely isolated mSMG cells were loaded with DCFH-DA ROS dye at 37°C for 30 min. Then, cells were stimulated with 50 μM of the three polyphenols and the vehicle control for 30 min, then 1 mM H2O2 was added, and cells were incubated at 37°C for 30 min. The cell suspension was added in triplicate to a 96-well clear-bottomed black plate, and fluorescence was measured at 490 nm excitation and 535 nm emission.
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