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.

Protocols

References

This product has been referenced in:
  • Calamita P  et al. SBDS-Deficient Cells Have an Altered Homeostatic Equilibrium due to Translational Inefficiency Which Explains their Reduced Fitness and Provides a Logical Framework for Intervention. PLoS Genet 13:e1006552 (2017). Flow Cyt ; Human . Read more (PubMed: 28056084) »
  • Wang LJ  et al. Zinc Finger E-Box Binding Protein 2 (ZEB2) Suppress Apoptosis of Vascular Endothelial Cells Induced by High Glucose Through Mitogen-Activated Protein Kinases (MAPK) Pathway Activation. Med Sci Monit 23:2590-2598 (2017). Functional Studies . Read more (PubMed: 28551696) »

See all 59 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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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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Thank you for getting in touch with your question regarding ab113851 DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit.


Since this is a fluorescent assay, the range of detection and linearity will vary a lot from instrument to ...

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DCFDA to quickly asses ROS activity

Excellent Excellent 5/5 (Ease of Use)
Abreviews
This kit works very well. The DCFDA concentration needs to be adjusted based on the customized experimental conditions. I used 40 uM rather than 20.
Username

Dr. Francesco Elia Marino

Verified customer

Submitted Mar 04 2016

The reference http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730091/(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730091/) cites PMID 12072178 (http://www.ncbi.nlm.nih.gov/pubmed/12072178) which apparently indicates that DCF is membrane permeable.

Thank you for your telephone enquiry yesterday.


I contact the laboratories. I am sorry we don't provide TBHP separately.

You could source this from an alternative supplier as either tert butyl hydrogen peroxide or hydrogen peroxi...

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1. The buffer provided is HBSS but you can use your own PBS in its place.
2. The number of cells per sample is difficult to generalize as it depends on instrument sensitivity, size of cells, etc.

Endogenous ROS Detection in Breast Cancer Cells

Good Excellent 5/5 (Ease of Use)
Abreviews
We used this compound (DCFDA) to detect downregulation of ROS by a novel antioxidant. Specifically we treated MDA-MB-231 breast cancer cells (adherent) with the antioxidant compound over night, then washed it out, subsequently added DCFDA, incubated for 4 hrs (longer is not recommended by abcam). Cells were imaged every 2 hrs (cells from separate wells of a well plate for each time point) under a fluorescent microscope. Overall the DCFDA levels were lower in antioxidant-treated cells than control, non-treated cells. In addition when we treated cells with H2O2, DCFDA levels were higher than control.
One problem with using DCFDA is that the fluorescent intensity seems to vary during imaging, as if the dye is unstable.
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Abcam user community

Verified customer

Submitted Sep 03 2014

Product works well for 24 hour activation of microglia

Excellent Excellent 5/5 (Ease of Use)
Abreviews
We are currently using the product to measure microglial activation after 24 hours in response to activating stimuli. The product has been giving us very consistent results and is very easy to use. The procedure is similar to one recommended in the FAQ section of the handbook.
Username

Neal Bennett

Verified customer

Submitted Aug 20 2014

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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