DCFDA / H2DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit (ab113851)

Overview

  • Product name
    DCFDA / H2DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit
    See all Oxidative Stress kits
  • Detection method
    Fluorescent
  • Assay type
    Cell-based (quantitative)
  • Product overview

    DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit (ab113851) uses the cell permeant reagent 2’,7’ –dichlorofluorescin diacetate (DCFDA, also known as H2DCFDA), a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell. After diffusion in to the cell, DCFDA / H2DCFDA 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.

  • Notes

    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.

  • Tested applications
    Suitable for: Flow Cyt, Functional Studiesmore details
  • Platform
    Reagents

Properties

Applications

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.

Application Abreviews Notes
Flow Cyt Use at an assay dependent concentration.
Functional Studies Use at an assay dependent concentration.

Images

  • Kobashigawa et al. (Pubmed 25127116) used the DCFDA ROS assay ab113851 to investigate the causes of the protective effects of metformin (Met) treatment in Doxorubicin (Dox) induced cardiotoxicity.

    They identified that in metformin treated H9c2 rat immortalized cardiomyoblasts, Met treatment reduced ROS levels induced by Dox (A). Values represent mean ± S.D. (n = 4).

    In combination with other assays, they developed the hypothesis that Dox induces increased ROS expression, leading to increased calcium levels and cell death, and that Met reduces this effect by increasing AMPK expression.

  • 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.

  • Labeled HL60 cells were treated with idarubicin or doxorubicin for 4 hours at multiple doses according to the protocol. At the end of the treatment cells were read end point in a fluorescent plate reader (Perking Elmer-Wallac 1420 Victor 2 Multilabel plate reader). Mean +/- standard deviation is plotted for 3 replicates from each condition. The dotted line represents the mean of 24 replicates of HL60 cells treated with 0.5% DMSO.

Protocols

References

This product has been referenced in:

See all 104 Publications for this product

Customer reviews and Q&As

In the protocol we recommend to run the assay in the absence of phenol red as it can increase the background. The background seems to be more of a problem on spectrophotometers than on flow cytometers.

Because it is well known that phenol ...

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I would suggest starting with 50 µM of TBHP as a positive control as we have observed linearity of signal when seeding Jurkat cells at 200,000 cells per well with treatments of TBHP from 50 – 500uM.

The amount of TBHP to use will h...

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In this case ter-Buytyl hydroperoxide solution is made up in 5M decane. 5mM decane could be used to treat the control culture, as the TBPH is given at 1000X concentration.

A measurement at 570nm will not detect the dye (DCFDA). The dye has the emission maximum at 529nm. We give 535nm, as the old plate reader with UV lamp can measure this wavelength and this works with the kit. I therefore propose that you buy the kit on...

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The lab says fixation will cause the dyes to leak from the cells. I think the assumption is that fixation may damage cell membranes. I did find a paper which describes fixation using methanol or acetic acid, after staining with a DCFDA derivative. DCFD...

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The lab has informed me that the cells must be alive, so fixed cells will not work with this kit.

In our experience, running the flow cytometer with cells in media did not appreciably affect the data – there was marginally less “background” if the cells were washed, but the slight improvement in data signal did not warrant the ext...

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1) The plate should be read with buffer (100uL/well). Cells must be alive during the reading.

2) For a plate reader assay, it is preferable not to use phenol red as there could be background. If you need to read to phenol red, make sure to i...

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Some types of tissue cultue cells adhere only weakly to plastic or glass and are easily washed away with buffer washes as you describe. Some things to try:

1. Make sure to was the cells very gently. Use a multi-channel pipettor to gently add ...

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The primary difference between white and black plates is their reflective properties. White plates reflect light and will maximize light output signal, black plates absorb light and reduce background and crosstalk. For this reason, white plates are com...

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1-10 of 42 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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