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DCFDA Assay Kit ab113851 includes DCFDA reagent, buffer, TBHP positive control, and a full protocol, for a fast way to set up a DCFDA assay on live cells. DCFDA taken up by cells is oxidized by reactive oxygen species with fluorescent readout (Ex/Em 485/535 nm) by microscopy, flow cytometer or plate reader.


Images

Fluorescence Microscopy - DCFDA / H2DCFDA - Cellular ROS Assay Kit (AB113851), expandable thumbnail
  • Cellular Activity - DCFDA / H2DCFDA - Cellular ROS Assay Kit (AB113851), expandable thumbnail
  • Fluorescence Microscopy - DCFDA / H2DCFDA - Cellular ROS Assay Kit (AB113851), expandable thumbnail
  • Cellular Activity - DCFDA / H2DCFDA - Cellular ROS Assay Kit (AB113851), expandable thumbnail
  • Functional Studies - DCFDA / H2DCFDA - Cellular ROS Assay Kit (AB113851), expandable thumbnail

Publications

Key facts

Detection method

Fluorescent

Sample types

Suspension cells, Adherent cells

Assay type

Cell-based (quantitative)

Assay time

40m

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Targets

Benzoic acid, 2-[3,6-bis(acetyloxy)-2,7-dichloro-9H-xanthen-9-yl]-

What's included?

300 Test
Components
10X Dilution Buffer (Sterile)
1 x 10 mL
20 mM DCFDA (Label)
1 x 35 µL
55mM TBHP
1 x 50 µL

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DCFDA Assay Kit ab113851 includes DCFDA reagent, buffer, TBHP positive control, and a full protocol, for a fast way to set up a DCFDA assay on live cells. DCFDA taken up by cells is oxidized by reactive oxygen species with fluorescent readout (Ex/Em 485/535 nm) by microscopy, flow cytometer or plate reader.

Key facts

Detection method

Fluorescent

Sample types

Suspension cells, Adherent cells

Assay type

Cell-based (quantitative)

Assay time

40m

Assay Platform

Microplate reader, Fluor. microscope, Flow cyt.

Storage

Shipped at conditions

Blue Ice

Appropriate long-term storage conditions

-20°C

Storage information

-20°C

Notes

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 semi 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 Carboxy-DCFDA N-succinimidyl ester, Fluorogenic esterase substrate ab145286 (Carboxy-DCFDA N-succinimidyl ester) and DCFA (5(6)-Carboxy-2',7'-dichlorofluorescein), green fluorophore 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.

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

Supplementary info

This supplementary information is collated from multiple sources and compiled automatically.

Activity summary

Oxidative stress refers to the imbalance between reactive oxygen species (ROS) and antioxidant defenses. Commonly studied alternate names include ROS and oxidative damage. ROS are highly reactive molecules that can damage proteins lipids and DNA. These molecules include free radicals like superoxide and non-radicals like hydrogen peroxide. ROS are expressed in various cellular compartments such as mitochondria the endoplasmic reticulum and peroxisomes. Oxidative stress markers like advanced oxidation protein products (AOPP) and assays like DCFH-DA and DCFDA (also known as DCFH and DCFH-DA assay) are commonly used for detection. The molecular weight of DCFH-DA is approximately 487.29 g/mol.

Biological function summary

Oxidative stress impacts various cellular processes and pathways. It does not function as a single protein but emerges from a complex interplay of biochemical reactions. These reactions contribute to cellular signaling regulation of gene expression and apoptosis. For instance H2DCFDA a derivative of DCFH-DA is important for measuring intracellular ROS levels. When cells metabolize H2DCFDA it fluoresces upon reacting with ROS indicating oxidative stress within cells. Deep red fluorescence emerges as a marker of increased ROS activity within certain experimental setups.

Pathways

Oxidative stress plays a significant role in the redox signaling pathway and the mitochondrial apoptosis pathway. In redox signaling oxidative stress influences the function of transcription factors like NF-kB and proteins like Nrf2 which regulate antioxidant response elements. In the mitochondrial apoptosis pathway oxidative stress leads to the release of cytochrome c which triggers the cascade leading to cell death. Both pathways illustrate how oxidative stress can influence life-or-death decisions within cells ultimately impacting cellular health and function.

Associated diseases and disorders

Oxidative stress links closely to neurodegenerative diseases such as Alzheimer's and cardiovascular diseases. In Alzheimer's for example oxidative stress can exacerbate the aggregation of amyloid-beta a protein associated with the disease. In cardiovascular diseases proteins like nitric oxide synthase (NOS) relate to oxidative stress where the imbalance can lead to oxidative damage in blood vessels contributing to atherosclerosis. The accumulation of oxidative stress can act as both a cause and a consequence in these pathological states indicating its dual role in disease processes.

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8 product images

  • Fluorescence Microscopy - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851), expandable thumbnail
    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/

    Fluorescence Microscopy - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851)

    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.

  • Cellular Activity - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851), expandable thumbnail
    Kobashigawa et al. (Pubmed 28056084)

    Cellular Activity - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851)

    DCFDA ROS assay used to study Doxorubicin cardiotoxicity.

    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.

  • Fluorescence Microscopy - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851), expandable thumbnail
    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/

    Fluorescence Microscopy - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851)

    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. Increased ROS generation in HCECs challenged with C. albicans and inhibition by the p38 activation inhibitor SB203580.

  • Cellular Activity - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851), expandable thumbnail
    This image is courtesy of an anonymous customer review

    Cellular Activity - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851)

    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.

  • Functional Studies - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851), expandable thumbnail

    Functional Studies - DCFDA / H2DCFDA - Cellular ROS 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.

  • Functional Studies - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851), expandable thumbnail

    Functional Studies - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851)

    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.

  • Functional Studies - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851), expandable thumbnail
    Image 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/

    Functional Studies - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851)

    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.

  • Flow Cytometry - DCFDA / H2DCFDA - Cellular ROS Assay Kit (ab113851), expandable thumbnail

    Flow Cytometry - DCFDA / H2DCFDA - Cellular ROS 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.

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