Reactive oxygen species (ROS) were measured using the cell permeant reagent 2′,7′‐dichlorofluorescin diacetate (DCFDA) according to the manufacturer's protocol (Abcam, ab113851). DCFDA is deacetylated by cellular esterases and oxidized by ROS into a highly fluorescent compound which was measured using a fluorescence microplate reader (excitation/emission wavelength of 488/535 nm). Amplex® Red reagent (10‐acetyl‐3,7‐dihydroxyphenoxazine) was used to detect hydrogen peroxide (H2O2). In the presence of peroxidase, the Amplex® Red reagent reacts with H2O2 to produce the red‐fluorescent oxidation product, resorufin, which was measured using fluorescence microplate reader (excitation/emission wavelength of 430/590 nm), according to the manufacturer's protocol (A22188, Invitrogen). To detect production of mitochondrial superoxide radical in live cells, the Mitochondrial Superoxide Detection Kit (ab219943, Abcam) was used, according to the manufacturer's protocols. Superoxide was measured using fluorescence microplate reader (excitation/emission wavelength of 540/590 nm).
A22188
Manufactured by Thermo Fisher Scientific
The A22188 is a laboratory centrifuge capable of separating samples based on their density and size. It provides high-speed rotation to generate the necessary centrifugal force for efficient sample separation.
Automatically generated - may contain errors
2 protocols using a22188
1
Measurement of Cellular ROS and H2O2
2
Amplex Red Assay for H2O2 Quantification
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The Amplex Red fluorescent probe activated by horseradish peroxidase (HRP) was used for H 2 O 2 detection. The experiment was performed according to the manufacturer's instructions (A22188, Invitrogen). In short, the reaction mixture containing 50 mM Amplex Red and 0.1 U/ml HRP was prepared in HBSS buffer. 100 mL/well of the reaction mixture were pipetted into 96-well plate and warmed at 37 C for 10 min. 20 mL of 2*10 4 cells in HBSS buffer were added to the well, and the fluorescence (excitation: 545 nm; emission 590 nm) were recorded by Spectra Max i3x plate reader (Molecular Devices).
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