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DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (11)Q&A (38)References (553)

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Effect of anethole on excessive ROS generation in hMSCs.
  • DCFDA ROS assay used to study Doxorubicin cardiotoxicity
  • Flow Cytometry - DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit  (ab113851)
  • p38 MAPK pathway involved in oxidative injury to HCECs challenged with C. albicans.
  • AbReview
  • Functional Studies - DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit  (ab113851)
  • Acute Effect of anthracyclines on ROS production in HL60 cells.
  • Reactive oxygen species (ROS) measured using the DCFDA assay

Key features and details

  • Assay type: Cell-based (quantitative)
  • Detection method: Fluorescent
  • Platform: Microplate reader, Fluor. microscope, Flow cyt.
  • Assay time: 40 min
  • Sample type: Adherent cells, Suspension cells

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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, DCFH-DA, and DCFH) to quantitatively assess reactive oxygen species in live cell samples.


    DCFDA / H2DCFDA / DCFH-DA / DCFH is a fluorogenic dye that measures hydroxyl, peroxyl and other reactive oxygen species (ROS) activity within the cell. NB: DCFDA and DCFA are also available as free molecules as ab145286 (Carboxy-DCFDA N-succinimidyl ester) and ab145439 (5(6)-Carboxy-2',7'-dichlorofluorescein).


    The DCFDA assay protocol is based on the diffusion of DCFDA / H2DCFDA / DCFH-DA / DCFH into the cell. It is then deacetylated by cellular esterases to a non-fluorescent compound, which is later oxidized by ROS into 2’, 7’ –dichlorofluorescein (DCF). DCF is highly fluorescent and is detected by fluorescence spectroscopy with excitation / emission at 485 nm / 535 nm.


    Chinese protocol available. See Protocols section below.


    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


    DCFDA assay protocol / ROS assay protocol summary (fluorescent microscopy):
    - wash adherent cells with buffer
    - stain with DCFDA for 45 min
    - wash in buffer
    - analyze with fluorescent microscope
    - maintain low light conditions to reduce photo-bleaching

  • 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. The number of measurements in microscopy is dependent on experimental setup.

    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.

    For reactive oxygen species, the most popular live cell ROS assay is DCFDA Assay ab113851 (green). Alternative ROS assays are available in orange (ab186028), red (ab186027), and deep red (ab186029).  ab238535 is used to measure ROS in biofluids, culture supernatants and cell lysates.

    For assays designed to differentiate ROS, superoxides, and reactive nitrogen species: to assay ROS and superoxides use ab139476; to assay ROS, superoxides, and reactive nitrogen species use ab139473; to assay superoxides use ab219943.

  • Platform

    Microplate reader, Fluor. microscope, Flow cyt.

Properties

  • Storage instructions

    Store at -20°C. Please refer to protocols.
  • Components 300 tests
    10X Dilution Buffer (Sterile) 1 x 10ml
    20 mM DCFDA (Label) 1 x 35µl
    55mM TBHP 1 x 50µl
  • Research areas

    • Kits/ Lysates/ Other
    • Kits
    • Cell Damage Kits
    • Oxidative stress
    • Metabolism
    • Pathways and Processes
    • Redox metabolism
    • Oxidative stress
    • Kits/ Lysates/ Other
    • Kits
    • Cell Metabolism Kits
    • Oxidative Stress Assay Kits
    • Oxidative Stress

Associated products

  • Related Products

    • DFCA DA (5(6)-Carboxy-2',7'-dichlorofluorescein diacetate), Fluorogenic esterase substrate (ab145415)
    • 10X Dilution Buffer (ab222287)

Images

  • Effect of anethole on excessive ROS generation in hMSCs.
    Effect of anethole on excessive ROS generation in hMSCs.Image from Rhee YH et al., BMC Cell Biol, 2018, 19: 12, Fig. 3B.; doi: 10.1186/s12860-018-0163-2 Reproduced under the Creative Commons license https://creativecommons.org/licenses/by/4.0/

    Effect of anethole on excessive ROS generation in hMSCs. hMSCs were exposed at 2 mM H2O2 for 30 min and incubated for 2 days in presence or absence of 50 μM anethole. ROS was measured by staining the cells with DCFDA cellular ROS detection assay kit according to the manufacturer’s instructions. ROS generation was observed under a fluorescence microscope at 200× magnification.

  • DCFDA ROS assay used to study Doxorubicin cardiotoxicity
    DCFDA ROS assay used to study Doxorubicin cardiotoxicityKobashigawa et al. (Pubmed 28056084)

    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.

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

    ab113851 (DCFDA) labeled and unlabeled Jurkat cells were treated with 50 µM tert-butyl Hydrogen Peroxide (tbHP), then analyzed by flow cytometry.

  • p38 MAPK pathway involved in oxidative injury to HCECs challenged with C. albicans.
    p38 MAPK pathway involved in oxidative injury to HCECs challenged with C. albicans.Image from Hua X et al., Sci Rep, 2017, 7: 10421, Fig. 4A.; doi: 10.1038/s41598-017-09636-w Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    p38 MAPK pathway involved in oxidative injury to HCECs challenged with C. albicans. Increased ROS generation in HCECs challenged with C. albicans and inhibition by the p38 activation inhibitor SB203580. 

  • AbReview
    AbReviewThis image is courtesy of an anonymous Abreview

    ROS levels analysis in human immortalized cardiomyocytes cells with ab113851 under different treatment conditions.

    Data are mean ± SEM of three different experiments. H2O2 800μM was used as Positive Control. The values of fluorescence intensity at each time point are indicated as the ratio of the value at specific t-time point on the value at time point zero (first measurement) (t-time point/t0). Briefly, cells were plated (seeding density 2.5 x 104 cells/cm2) in FBS-supplemented medium w/o phenol red onto a 96 black well plate. After 24 hours the cells were washed one time with 1X buffer (provided in the kit), then the cells were incubated with DFCDA 10 μM for 30 min at 37° C protected from light. Following incubation the wells were washed with PBS and the cells were exposed to treatments of interest in FBS-supplemented medium w/o phenol red. ROS production was determined immediately by measuring the formation of fluorescent dichloro fluorescein (DCF), using a PerkinElmer VICTOR3, at an Ex-485 and Em-535 nm. Measurements were done every 30 min for six hours. The value of fluorescence intensity at each time point is reported. The value reported was obtained by the ratio of fluorescence at specific time point on fluorescence at time 0, which was measured immediately after DCFDA incubation.

    See Abreview

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

    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.

  • Acute Effect of anthracyclines on ROS production in HL60 cells.
    Acute Effect of anthracyclines on ROS production in HL60 cells.

    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.

  • Reactive oxygen species (ROS) measured using the DCFDA assay
    Reactive oxygen species (ROS) measured using the DCFDA assayImage from Davidson et al., Sci Rep, 2018. Fig. 2B.; doi: 10.1038/s41598-018-35455-8 Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

    Reactive oxygen species (ROS) measured using the DCFDA assay in human primary articular chondrocytes. Cells were treated with 100 µM tert-butyl-hydroperoxide (tBHP) alone (4 h) ± pre-treatment with apigenin.

Protocols

  • Protocol Booklet

Click here to view the general protocols

Datasheets and documents

  • SDS download

  • Datasheet download

    Download

References (553)

Publishing research using ab113851? Please let us know so that we can cite the reference in this datasheet.

ab113851 has been referenced in 553 publications.

  • Wei F  et al. A novel approach for the prevention of ionizing radiation-induced bone loss using a designer multifunctional cerium oxide nanozyme. Bioact Mater 21:547-565 (2023). PubMed: 36185749
  • Ansari MSZ  et al. Pharmacological targeting of CBP/p300 drives a redox/autophagy axis leading to senescence-induced growth arrest in non-small cell lung cancer cells. Cancer Gene Ther N/A:N/A (2022). PubMed: 36117234
  • Dieter F  et al. Redox Active a-Lipoic Acid Differentially Improves Mitochondrial Dysfunction in a Cellular Model of Alzheimer and Its Control Cells. Int J Mol Sci 23:N/A (2022). PubMed: 36012451
  • Wang C  et al. Protective effect of the curcumin-baicalein combination against macrovascular changes in diabetic angiopathy. Front Endocrinol (Lausanne) 13:953305 (2022). PubMed: 36060932
  • Alipour S  et al. Alantolactone and ZnO nanoparticles induce apoptosis activity of cisplatin in an ovarian cancer cell line (SKOV3). Res Pharm Sci 17:294-304 (2022). PubMed: 35531132
View all Publications for this product

Customer reviews and Q&As

Show All Reviews Q&A
Submit a review Submit a question

1-10 of 49 Abreviews or Q&A

Question

I see media with phenol red has a higher background in this assay. What is the typical background OD seen with phenol red media?

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Abcam community

Verified customer

Asked on Mar 21 2014

Answer

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 red gives reading at 530nm we do not have development data using this kit in the presence of phenol red.

Read More

Jeremy Kasanov

Abcam Scientific Support

Answered on Mar 21 2014

Question

ab113851 provides 55mM TBHP. What concentration of TBHP do you suggest to use as positive control on A375 cell line (an adherent cell line)?
In your suggested assay templates figure 3 in your protocol booklet, it mentioned vehicle titration control or vehicle control. What is vehicle titration control or vehicle control?

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Abcam community

Verified customer

Asked on Sep 04 2013

Answer

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 however depend on the sensitivity of the cell line to TBHP (HL60 and Jurkat cells are very sensitive whereas HepG2 are very insensitive). Therefore you may have to optimise this concentration depending on your cells sensitivity to TBHP.

The vehicle control or vehicle titration refers to the diluent used in the test compounds. Some researchers dilute their compounds in ethanol or DMSO. If the test compounds are assayed in titration, we suggest for the vehicle or diluent of the test compound to be also assayed in titration. The vehicle/diluent is simply a negative control. It simply shows that it is only the compound TBHP that is responsible for the increase in ROS and not some other confounding variable such as the diluent itself

Read More

Elisa Thomas

Abcam Scientific Support

Answered on Sep 04 2013

Question

What is the vehicle that is used as a negative control treatment for cells instead of treating with tbHP?

Read More

Abcam community

Verified customer

Asked on Jul 25 2013

Answer

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.

Read More

Jeremy Kasanov

Abcam Scientific Support

Answered on Jul 25 2013

Question

Is measurement at 570 nm possible?

Read More

Abcam community

Verified customer

Asked on May 09 2013

Answer

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 only, if you find a plate reader capable of reading at 535 or 529nm.

Read More

Abcam Scientific Support

Answered on May 09 2013

Question

My sample is moving cells - spermatozoa. So it is not easy to take a good picture without fixing the sample. Are the three dyes compatible with fixation of the cells (i.e, 4% PFA or other)? Would the microscopic observation work with fixed cells to reflect the changes of ROS (or RNS) levels?

Read More

Abcam community

Verified customer

Asked on Apr 19 2013

Answer

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. DCFDA is the reagent that stains ROS in the kit ab113851, and I suspect that it is the green dye in the other two kits, the ROS/NOS and superoxide assays, though our source for those kits has not confirmed. A modified fixed staining method for the simultaneous measurement of reactive oxygen species and oxidative responses. Shen et al, 2013. PMID: 23178299 http://www.ncbi.nlm.nih.gov/pubmed/23178299

Read More

Abcam Scientific Support

Answered on Apr 19 2013

Question

I want to try this kit for confocal microscopy, do you think it will work with cells fixed with formaldehyde?

Read More

Abcam community

Verified customer

Asked on Apr 17 2013

Answer

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

Read More

Abcam Scientific Support

Answered on Apr 17 2013

Question

Why is the staining of cells before treatment? And why does it indicate not to wash the cells after treatment for the Flow Cytometry protocol, when it allows for washing with the microplate protocol? Won't the media from my cell treatment affect my flow cytometry reading?

Read More

Abcam community

Verified customer

Asked on Feb 27 2013

Answer

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 extra steps. Hence we recommend the simpler procedure of reading directly in media.

Staining of the cells before treatment allows (1) bulk labeling of the cells with DCFDA (2) the compounds/treatments to remain on the cells at the time of the reading. For example (with drug treatments), if treatments were done first, then subsequent addition of DCFDA would lead to a dilution of the treatments (unless drugs were also spiked into the DCFDA solution). Again, this is a case of protocol simplification. Also note that for some drug treatments the effect on ROS levels can be rapidly reversed e.g. a drug can induce ROS levels in a cell, but cellular ROS levels can decrease rapidly as soon as the drug is removed/diluted.

Read More

Abcam Scientific Support

Answered on Feb 27 2013

Question

On pages 7-9 of the protocol:
1) After the wash, should more buffer be added to the wells before reading or are they supposed to be read empty? If buffer should be added, how much?
2) For treated adherent cells (page 8 step 6), can the compounds be diluted in media with phenol red?
3) If I do dilute the compounds in media instead of 1x buffer, do I need to wash the wells and add 1x buffer prior to reading, for consistency with the untreated controls?

Read More

Abcam community

Verified customer

Asked on Dec 06 2012

Answer

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 include appropriate negative controls = (1) unstained treated cells with media, (2) media only with no cells."


3) It is essential to read in the presence of compounds. If you need to read in media ensure that untreated controls are also read in media and as in answer #2 make sure you include appropriate negative controls to take into account any background signal.

Read More

Abcam Scientific Support

Answered on Dec 06 2012

Question

When I washed the cells with PBS, I didn't see that any cells were washed away. However, after I stained with DCFDA, the cells were washed off the 96 well plate with 1*buffer solution. Have other customers experienced this? Do you have any suggestions to fix the issue?

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Abcam community

Verified customer

Asked on Oct 22 2012

Answer

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 was buffer by letting it slowly stream down the side of each well.

2. Alternatively, you can coat the plates to increase cell adhernce. Poly-L-lysine is often used to coat wells for this purpose. Alternative treatments for coating the plastic wells may exist which are better for your application however.

Read More

Abcam Scientific Support

Answered on Oct 22 2012

Question



In the product instruction, it says I should seed the cells in sterile, tissue culture treated, BLACK 96-well microplates, can I use sterile, tissue culture treated, White 96-well microplates instead? What is the difference between them?

For adherent cells, at the last step before reading, it says "Wash the plate once with 1X buffer solution" and nothing else. Does it mean when read in the plate reader, there is no liquid in the well? Will it affect the result? Or I just need to keep the buffer solution in the well?

Read More

Abcam community

Verified customer

Asked on Oct 22 2012

Answer

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 commonly used for luminescent assays and black plates are used for fluorescent assays. For this reason we recommend black plates with this kit. Thus white plates may result in higher crosstalk and higher background with this kit.

Regarding second question, if toxicity assay is not of interest, just add 100 uL/well of 1Xsupplemented buffer solution after the washing step.

Read More

Abcam Scientific Support

Answered on Oct 22 2012

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