MitoSciences (ms958)

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

Overview

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

    ab113851 contains sufficient materials for approximately 300 measurements in microplate format and 70 measurements (35 mL) by flow cytometry.

     

    Abcam's DCFDA - Cellular Reactive Oxygen Species Detection Assay kit 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.

     

     

  • 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

Properties

  • Alternative names
    • MS958

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.

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.

Protocols

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

This product has been referenced in:

See all 20 Publications for this product

Product Wall

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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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.

For adherent cells (e.g. HeLa) we reco...

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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.
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neal bennett

Verified customer

Submitted Aug 20 2014

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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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 ...

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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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In this case ter-Buytyl hydroperoxide solution is made up in 5M decane.

The customer could use 5M decane and treat the control culture with 5mM decane as the TBPH is given at 1000X concentration. Short treatments are a must here.

The TBHP is tert-butyl Hydrogen Peroxide.



I have to following suggestions:

1. It should be possible to use a cell viability assay after reading ROS, since the cells will be alive. Our lab has not tried a particular stain after DCFDA staining but it might be worth a try. Read More

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