This product (DCFH-DA) is cell-permeable and converts intracellularly to the fluorescent DCF upon ROS oxidation, enabling ROS detection in EGFP-transfected cells. However, direct fluorescence in the GFP channel is not ideal due to spectral overlap between DCF (Ex ~504 nm, Em ~529 nm) and EGFP (Ex ~488 nm, Em ~507–509 nm), which can interfere with signal and ROS quantification. If EGFP-transfected cells are used, include controls such as EGFP-only cells, DCFH-DA-only cells, unstained cells, and a positive ROS control. Alternatively, consider a red/far-red ROS probe to avoid spectral overlap.
D6883
2′,7′-Dichlorodihydrofluorescein diacetate
≥97% (HPLC), powder, cell-permeable non-fluorescent probe
Synonym(s):
2′,7′-Dichlorofluorescin diacetate, DCFH-DA, 2′,7′-Dichlorodihydrofluorescein diacetate
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About This Item
Empirical Formula (Hill Notation):
C24H16Cl2O7
CAS Number:
Molecular Weight:
487.29
NACRES:
NA.77
PubChem Substance ID:
UNSPSC Code:
12352200
MDL number:
Beilstein/REAXYS Number:
4610273
Assay:
≥97%
Form:
powder
Pricing and availability is not currently available.
Product Name
2′,7′-Dichlorodihydrofluorescein diacetate, ≥97%
Quality Segment
assay
≥97%
form
powder
storage temp.
−20°C
SMILES string
CC(=O)Oc1cc2Oc3cc(OC(C)=O)c(Cl)cc3C(c2cc1Cl)c4ccccc4C(O)=O
InChI
1S/C24H16Cl2O7/c1-11(27)31-21-9-19-15(7-17(21)25)23(13-5-3-4-6-14(13)24(29)30)16-8-18(26)22(32-12(2)28)10-20(16)33-19/h3-10,23H,1-2H3,(H,29,30)
InChI key
PXEZTIWVRVSYOK-UHFFFAOYSA-N
General description
2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) is an oxidation-sensitive fluorescent dye used to detect the intracellular reactive oxygen species (ROS) level. DCFH-DA is a cell-permeable non-fluorescent probe, that can be hydrolyzed by intracellular esterase to DCFH, which forms fluorescent DCF when oxidized by the intracellular ROS.
The excitation wavelength is 504 nm and emission wavelength is 529 nm in ethanol. The use of DMSO as the solvent has not been determined, however various publications suggest that it is suitable.
Application
2′,7′-Dichlorofluorescin diacetate is suitable for use in:
- sensitive and rapid quantitation of oxygen-reactive species in response to oxidative metabolism
- microplate assay for detecting oxidative products in phagocytic cells
- quantitative multiwell myeloid differentiation assay
Applications include sensitive and rapid quantitation of oxygen-reactive species in response to oxidative metabolism; microplate assay for detecting oxidative products in phagocytic cells, and quantitative multiwell myeloid differentiation assay.
Preparation Note
This product should be stored in powdered format at -20°C.
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This Item | |||
|---|---|---|---|
| description ≥97% | description Cell-permeable fluorogenic probe that is useful for the detection of reactive oxygen species (ROS) and nitric oxide (•NO) and for the determination of the degree of overall oxidative stress. | description suitable for use as an indicator in chloride titration, ~90% (TLC), powder | description ACS reagent |
| form powder | form crystalline solid | form powder | form powder |
| assay ≥97% | assay ≥94% (HPLC) | assay ~90% (TLC) | assay - |
| Quality Level 200 | Quality Level 100 | Quality Level 200 | Quality Level 200 |
| storage temp. −20°C | storage temp. −20°C | storage temp. room temp | storage temp. room temp |
Storage Class
11 - Combustible Solids
wgk
WGK 3
ppe
Eyeshields, Gloves, type N95 (US)
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Is this product compatible with EGFP transfected cells?
1 answer-
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Can you suggest a protocol for detection of ROS in colon tissue from rat?
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This product is not tested specifically for in vitro or in vivo use. A protocol is not currently available. However, a search of the literature reveals several publications that may be helpful. Please see the links below to review examples:
A method to determine reactive oxygen species production in intestinal and liver cell cultures using the 2’,7’-dichlorodihydrofluorescein diacetate assay
https://www.sciencedirect.com/science/article/pii/S2215016124000694Detection of Total Reactive Oxygen Species in Adherent Cells by 2’,7’-Dichlorodihydrofluorescein Diacetate Staining
https://pmc.ncbi.nlm.nih.gov/articles/PMC7712457/Helpful?
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Is the product compatible with RFP transfected cells?
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Yes, RFP (Red Fluorescent Protein) and DCFH (2′,7′-Dichlorodihydrofluorescein) are spectrally compatible and can be used together for multicolor imaging or flow cytometry.
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Hi, is the product suitable for ROS detection in a cell-free system? If not, could you please recommend a DCFH related product that works in a system without cells? Thanks
1 answer-
This product as supplied is DCFH-DA and used for intracellular ROS detection. Please see the link below for understanding this process.
https://pubs.acs.org/doi/10.1021/acs.analchem.7b00043For use in a cell-free system, deacetylation of DCFH-DA into DCFH must be performed before use.
Please see the link below for this method.
https://www.sciencedirect.com/science/article/pii/S0006295203000832?via%3DihubHelpful?
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Is D6883 2´,7´-Dichlorofluorescin diacetate suitable for flow cytometry analyses?
1 answer-
While the specifications for this probe do not include assaying for specific applications, there are many publications showing its use in flow cytometry applications:
https://www.sciencedirect.com/science/article/pii/S0022030215000909
https://iubmb.onlinelibrary.wiley.com/doi/pdf/10.1002/iub.1895
https://www.tandfonline.com/doi/pdf/10.1080/21655979.2021.1994737
https://journal.waocp.org/article_91422_6ccd2a19eb9159073edeb8f9ce0be660.pdf
https://pmc.ncbi.nlm.nih.gov/articles/PMC7842666/Helpful?
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What is the protocol for measuring ROS using D6883? Specifically, what kind of media or buffer is used with this product?
1 answer-
The specific protocol for measuring ROS using D6883 and the type of media or buffer to be used with this product are not available. However, typically, this product is added to a buffer solution shortly before assessing the fluorescent signal. For further guidance, refer to the following citations for relevant information:
1. Novel Antioxidant Tripeptide "ACQ" Can Prevent UV-Induced Cell Death and Preserve the Number of Epidermal Stem Cells
PMID: 261996772. Detection of reactive oxygen species (ROS) by the oxidant-sensing probe 2',7'-dichlorodihydrofluorescein diacetate in the cyanobacterium Anabaena variabilis PCC 7937
PMID: 2057064These publications may provide insights into the protocol for using D6883 and the type of media or buffer used with this product.
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What concentration of DCF can I use for A549 cells?
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This product has not been tested for use in cell culture. A search of the literature suggests working concentrations range from 5 µM to 0.1 mM. See the link below to a publication that specifically looked at A549 cells using several dyes, including DCFDA:
https://pubs.rsc.org/en/content/articlehtml/2017/ra/c7ra01025gHelpful?
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What concentration of DCF can I use for THP-1 monocyte cell line?
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The optimal concentration of 2′,7′-Dichlorofluorescin diacetate (DCFDA) for use with THP-1 monocytes will need to be determined by the end-user. See the link below to a paper that reported using DCF at 40 uM with THP-1 cells.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8961584/Helpful?
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Is it possible to view this probe under a fluorescence microscope if the probe is solubilised in DMSO? What are the excitation and absorption wavelengths?
1 answer-
The excitation wavelength is 504 nm and the emission wavelength is 529 nm in ethanol. The use of DMSO as the solvent has not been determined, however, various publications suggest that it is suitable. See the link below for an example:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7712457/Helpful?
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Protocol please! Thank you very much!
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It may be helpful to view the following articles as there is not a specific protocol for this product:
Helpful?
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