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DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit (ab113851) uses the cell permeant reagent 2’,7’ –dichlorofluorescin diacetate (DCFDA), a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell. After diffusion in to the cell, DCFDA is deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2’, 7’ –dichlorofluorescein (DCF). DCF is a highly fluorescent compound which can be detected by fluorescence spectroscopy with maximum excitation and emission spectra of 495 nm and 529 nm respectively.
This kit contains sufficient materials for approximately 300 measurements in microplate format and 70 measurements (35 mL) by flow cytometry.
This kit is not compatible with fixed samples. Stained cells must be measured live.
Store all components at 4°C in the dark. The kits are stable for at least 6 months from receipt. For longer term storage, keep at -20°C to -80°C in the dark.
|Dilution Buffer (10X, sterile)||1 x 10ml|
|Label (20 mM DCFDA, 1000X)||1 x 35µl|
|TBHP (55 mM)||1 x 50µl|
Our Abpromise guarantee covers the use of ab113851 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Flow Cyt||Use at an assay dependent concentration.|
|Functional Studies||Use at an assay dependent concentration.|
ab113851 (DCFDA) labeled and unlabeled Jurkat cells were treated with 50 µM tert-butyl Hydrogen Peroxide (tbHP), then analyzed by flow cytometry.
Jurkat cells were labeled with DCFDA (20 µM) or unlabeled (none) and then cultured an additional 3 hours with or without 50 µM tert-butyl hydrogen peroxide (TBHP) according to the protocol. Cells were then analyzed on a fluorescent plate reader. Mean +/- standard deviation is plotted for 4 replicates from each condition. TBHP mimics ROS activity to oxidize DCFDA to fluorescent DCF.