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


  • Product nameDCFDA - Cellular Reactive Oxygen Species Detection Assay Kit
    See all Oxidative Stress kits
  • Detection methodFluorescent
  • Tests
    1 x 300 test
  • Assay typeCell-based (quantitative)
  • Product overview

    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.

  • 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 applicationsFlow Cyt, Functional Studiesmore details
  • PlatformReagents



    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.

    DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit images

    • 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.


    References for DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit (ab113851)

    This product has been referenced in:
    • Ye BH  et al. Preventive effect of gomisin J from Schisandra chinensis on angiotensin II-induced hypertension via an increased nitric oxide bioavailability. Hypertens Res 38:169-77 (2015). Functional Studies . Read more (PubMed: 25427681) »
    • Yang WH  et al. Imipramine blue halts head and neck cancer invasion through promoting F-box and leucine-rich repeat protein 14-mediated Twist1 degradation. Oncogene N/A:N/A (2015). Read more (PubMed: 26257063) »

    See all 28 Publications for this product

    Product Wall

    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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    DCFDA to quickly asses ROS activity

    Excellent Excellent 5/5 (Ease of Use)
    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.


    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)
    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.

    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)
    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.

    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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    We have not tested A375 cell line, but I’m confident that you could follow the steps in the protocol for adherent cells. This dye should work on any cell line. If cell lines other than those specified in the protocol are used, then you ...

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