Description
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.
Product details:
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
ab145286
(Carboxy-DCFDA N-succinimidyl ester) and
ab145439
(5(6)-Carboxy-2',7'-dichlorofluorescein).
How the assay works
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.
Assay protocol summary
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
How other researchers are using
DCFDA / H2DCFDA - Cellular ROS Assay Kit has been used in a variety of sample type including:
Cola rostrata K plant cells
Human monocyte cells THP-1
Mouse lung lysates
References:
1- Ajayi BE et al. 2023
2-Shrivastava G et al. 2024
3-Jiang Y et al. 2023
Properties and Storage Information:
Shipped at conditions-Blue Ice, Appropriate short-term storage conditions--20°C, Appropriate long-term storage conditions--20°C, Storage information--20°C
Supplementary Information:
This supplementary information is collated from multiple sources and compiled automatically.
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.
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.
