Overview

  • Product name
    DCFDA / H2DCFDA - Cellular ROS Assay Kit
    See all Oxidative Stress kits
  • Detection method
    Fluorescent
  • Sample type
    Adherent cells, Suspension cells
  • Assay type
    Cell-based (quantitative)
  • Assay time
    0h 40m
  • Product overview

    DCFDA - Cellular ROS Assay Kit / Reactive Oxygen Species 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 excitation / emission at 495 nm / 529 nm.


    DCFDA assay protocol / ROS assay protocol summary (microplate):
    - collect suspension cells in tube / seed and allow attachment of adherent cells in 96-well plate
    - wash in buffer
    - stain with DCFDA for 30 min (suspension) / 45 min (adherent), wash with buffer
    - if suspension cells, transfer to microplate
    - analyze with microplate reader


    DCFDA assay protocol / ROS assay protocol summary (flow cytometry):
    - collect cells in tubes
    - stain with DCFDA for 30 min (without washing)
    - analyze with flow cytometer

  • Notes

    Previously called DCFDA / H2DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit.

    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.


    Related products

    Review the oxidative stress marker and assay guide, or the full metabolism assay guide to learn about more assays for metabolites, metabolic enzymes, mitochondrial function, and oxidative stress, and also how to assay metabolic function in live cells using your plate reader.

  • Platform
    Microplate reader, Fluor. microscope, Flow cyt.

Properties

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:
  • Degl'Innocenti D  et al. Oxadiazon affects the expression and activity of aldehyde dehydrogenase and acylphosphatase in human striatal precursor cells: A possible role in neurotoxicity. Toxicology 411:110-121 (2019). Read more (PubMed: 30391265) »
  • Li Z  et al. APC-Cdh1 Regulates Neuronal Apoptosis Through Modulating Glycolysis and Pentose-Phosphate Pathway After Oxygen-Glucose Deprivation and Reperfusion. Cell Mol Neurobiol 39:123-135 (2019). Read more (PubMed: 30460429) »
See all 160 Publications for this product

Customer reviews and Q&As

Filter by Ratings

1-5 of 5 Abreviews

ROS detection in LPA treated microglia

Good Excellent 5/5 (Ease of Use)
Abreviews
The kit is easy to use and the results are quite constant. In our experiments, erum-starved primary cells were incubated for 45min with 20µM DCFDA, then treated for the indicated time points with 1µM LPA and the fluorescence intensity was measured using a Wallac Victor 1420 Multilabel Counter. tert-Butyl hydroperoxide (tBuOOH) was used as a positive control.

Dr. Joanna Plastira

Verified customer

Submitted Oct 11 2018

Abreviews
We tried to measure ROS release in RAW 264.7 cells using this kit. Used the kit for Microplate assay using 96-well plates. The protocol is very simple and easy to perform. After seeding cells into the plate for overnight, loaded the cells with DCFDA (25µM final concentration) for 45 min, before treating cells with our test compound. We also included TBHP, a positive control included in the kit, in our protocol. Cells were treated for 2-4h with our test compound. Unfortunately, we could not detect any increased signal in our treatment group compared to its non-treated group. Surprisingly, the positive control (TBHP) also did not show increased signal in our experiment. The results that we saw were unexpected and inconclusive. Although the protocol is very easy to perform, the kit did not work in our hands.

Abcam user community

Verified customer

Submitted Jun 04 2018

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.

Dr. Francesco Elia Marino

Verified customer

Submitted Mar 04 2016

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.

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.

Neal Bennett

Verified customer

Submitted Aug 20 2014

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

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