製品の概要

  • 製品名
    DCFDA / H2DCFDA - Cellular ROS Assay Kit
    Oxidative Stress キット 製品一覧
  • 検出方法
    Fluorescent
  • サンプルの種類
    Adherent cells, Suspension cells
  • アッセイタイプ
    Cell-based (quantitative)
  • 全工程の試験時間
    0h 40m
  • 製品の概要

    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

  • 特記事項

    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.

  • 試験プラットフォーム
    Microplate reader, Fluor. microscope, Flow cyt.

製品の特性

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  • 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.

プロトコール

参考文献

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 157 Publications for this product

レビューと Q&A

1-10 of 43 Abreviews or Q&A

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.

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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

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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.

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Question
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.

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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

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Answer

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

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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.

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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.

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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.

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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.

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1-10 of 43 Abreviews or Q&A

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

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