02 Oxidative Stress

ROS Assay Kit -Highly Sensitive DCFH-DA-

ROS Detection

High-sensitivity detection of total ROS
Detectable using fluorescence microscope, plate reader, and flow cytometer

Product code

R252　 ROS Assay Kit -Highly Sensitive DCFH-DA-

Unit size	Price	Item Code
100 tests		R252-10

Component

100 tests	・Highly Sensitive DCFH-DA Dye ・Loading Buffer (10x)	10 ×l×1 1.0 ml×1

C of A

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Description

Reactive oxygen species (ROS) are primarily produced in mitochondria during ATP synthesis. ROS play an essential role in signaling pathways and the immune system, whereas increasing ROS is associated with diseases and cellular senescence. Recent studies suggested that ferroptosis is a new type of cell death characterized by iron dependency and increasing ROS. Thus, ROS detection has been attracting considerable interest in ferroptosis research. DCFH-DA is widely used for ROS detection, but it has some limitations like weak fluorescence signals and high background. Dojindo’s ROS Assay Kit -Highly Sensitive DCFH-DA- overcomes these limitations. The dye employed in the kit allows ROS detection with higher sensitivity than DCFH-DA. Moreover, the Loading Buffer included in this kit maintains cellular health during assays.

Manual

Manual

Technical info

The selectivity of these probes for ROS
The reactivity of the Highly Sensitive DCFH-DA for ROS is similar to the reactivity of 2’-7’ dichlorofluorescein diacetate (DCFH-DA). The Highly Sensitive DCFH-DA also has similar fluorescence characteristics (λex: 505 nm, λem: 525 nm) to DCFH-DA. Therefore, ROS is detectable at the same excitation/fluorescence wavelength.

Comparison of detection sensitivity
Hydrogen peroxide (H₂O₂)-treated HeLa cells (1×10⁴ cells/ml) were stained with DCFH-DA or the ROS Assay Kit-Highly
Sensitive DCFH-DA, and the detectability of intracellular ROS was compared between two detection kits. As a result, the ROS Assay Kit-Highly Sensitive DCFH-DA in high-sensitivity detection of intracellular ROS was better than DCFH-DA.

① Detection using fluorescent microscope

Detection conditions
Ex. 488 nm / Em. 500 - 560 nm
Cell type：HeLa cells
（scale bar：50 µm）

② Detection using microplate reader

Detection conditions
Ex. 490 - 520 nm / Em. 510 - 540 nm
Cell type：HeLa cells

③ Detection using flow cytometer

Detection conditions
FITC laser gain：215 V
Cell type：HeLa cells

Detection of endogenous ROS in A549 cells treated with Elastin.

Elastin treatment, which inhibits cystine/glutamate antiporter (xCT), is known to induce iron-dependent cell death, also known as ferroptosis. The imaging of changes in intracellular ROS revealed that elastin treatment increased the generation of ROS in elastin-treated A549 cells.

（scale bar：50 µm）

Detection conditions
Intracellular ROS（Highly Sensitive DCFH-DA Dye）： Ex. 488 nm / Em. 500 - 560 nm

Potocol
1. A549 cells (1×10⁴ cells/ml) in DMEM (supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin) were seeded on ibidi 8-well plates.
2. The cells were cultured overnight in an incubator set at 37℃ and equilibrated with 95% air and 5% CO₂.
3. Medium was removed and DMEM containing Elastin (50 μmol/l) was added to the cells.
4. The cells were cultured overnight as in step 2.
5. After removing the supernatant, the cells were washed twice with HBSS and treated with Highly Sensitive DCFH-DA working solution.
6. The cells were incubated for 30 minutes as in step 2.
7. Supernatant was removed, and cells were washed twice with HBSS and refilled with HBSS. The cells were observed under a fluorescence microscope.

Detection of endogenous ROS in RAW264.7 cells treated with lipopolysaccharide (LPS).

In LPS-treated RAW264.7 cells, the imaging of changes in intracellular ROS revealed that LPS treatment increased the generation of ROS.

（scale bar：50 µm）

Detection conditions
Intracellular ROS（Highly Sensitive DCFH-DA Dye）： Ex. 488 nm / Em. 500 - 560 nm

Potocol
1. RAW264.7 (3×10⁴ cells/ml) in DMEM (supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin) were seeded on ibidi 8-well plates.
2. The cells were cultured overnight in an incubator set at 37℃ and equilibrated with 95% air and 5% CO₂.
3. Medium was removed and DMEM containing LPS (500 ng/ml) was added to the cells.
4. The cells were cultured for 20 hours as in step 2.
5. After removing the supernatant, the cells were washed twice with HBSS and treated with Highly Sensitive DCFH-DA working solution.
6. The cells were incubated for 30 minutes as in step 2.
7. Supernatant was removed, and cells were washed twice with HBSS and refilled with HBSS. The cells were observed under a fluorescence microscope.

Fluorescence Property

Comparison with Existing Reagents

A comparison of Dojindo Laboratories' probe and other probes for ROS detection.

	Dojindo Laboratories		Company T
Code	R252	R253	-	-
Product Name	ROS Assay Kit -Highly Sensitive DCFH-DA-	ROS Assay Kit Photo-oxidation Resistant DCFH-DA-	Probe D	Probe C
Photo-oxidation Resistant	- Without resistant ability	★★★★★ The highest resistant ability	- Without resistant ability	- Without resistant ability
Cell fixation	- Without retention ability	★★★★★ The highest retention ability	- Without retention ability	★★★ OK for fixation
Sensitivity (Intracellular)	★★★★★ The highest sensitivity	★★★★ Better sensitivity	★★★ Ordinary sensitivity	★★★ Ordinary sensitivity

References

Open References

No.	Sample	Instrument	Reference (Link)
1)	Cell U-251MG cells	Fluorescence microscope	S. Kato, "Effects of platinum‑coexisting dopamine with X‑ray irradiation upon human glioblastoma cell proliferation", Hum. Cell, 2021, doi:10.1007/s13577-021-00591-3.
2)	Cell Valve Interstitial Cells	Fluorescence microscope	K. Kanno, T. Sakaue, M. Hamaguchi, K. Namiguchi, D. Nanba, J. Aono, M. Kurata, J. Masumoto, S. Higashiyama, H. Izutani, "Hypoxic Culture Maintains Cell Growth of the Primary Human Valve Interstitial Cells with Stemness", Int. J. Mol. Sci. 2021, doi:10.3390/ijms221910534.
3)	Cell BPH-1 Cell (LPS induced)	Flow Cytometer	C. Fang, L. Wu, M. Zhao, T. Deng, J. Gu, X. Guo, C. Li, W. Li, X. Zeng, "Periodontitis Exacerbates Benign Prostatic Hyperplasia through Regulation of Oxidative Stress and Inflammation", Oxid. Med. Cell. Longevity, 2021, doi:10.1155/2021/2094665.
4)	Bacteria （Synechococcus elongatus PCC7942)	Plate Reader	M. Tamoi and S. Shigeoka, "CP12 Is Involved in Protection against High Light Intensity by Suppressing the ROS Generation in Synechococcus elongatus PCC7942", Plants, 2021, doi:10.3390/plants10071432.
5)	Cell SW982	Fluorescence microscope	S. Xia, Z. Ma, B. Wang, F. Gao, C. Yi, X. Zhou, S. Guo, L. Zhou, "In vitro anti-synovial sarcoma effect of diallyl trisulfide and mRNA profiling", Gene, 2021, doi:10.1016/j.gene.2021.146172.
6)	Cell BRL3A Cell	Fluorescence microscope	Z. Luo, Q. Gao, H. Zhang, Y. Zhang, S. Zhou, J. Zhang, W. Xu, J. Xu, " Microbe-derived antioxidants attenuate cobalt chloride-induced mitochondrial function, autophagy and BNIP3-dependent mitophagy pathways in BRL3A cells", 2022, doi:10.1016/j.ecoenv.2022.113219.

Q & A

Q Can you show me any example of a positive control?: A

Please refer to the technical manual for the example of detection using H₂O₂-treated HeLa cells.

Q May I use PBS instead of HBSS to wash cells after staining?: A

We recommend using HBSS to reduce cell damage. If you do not have HBSS, please rinse them with a culture medium.

Q May I use anything other than the Loading Buffer for the preparation of the Highly Sensitive DCFH-DA working solution?: A

To reduce cell damage resulting from staining, we recommend using the Loading Buffer supplied with the kit; however, you can also use Hanks’ HEPES or HBSS for the preparation. Please use a serum-free medium if you prepare the working solution in a culture medium.

Q Is there anything that I should be careful of when using a fluorescence microscope?: A

The Highly Sensitive DCFH-DA reacts with ROS and then becomes oxidized. Fluorescence is emitted after the oxidation of dye. When exposed to the excitation light for a long period, the dye becomes oxidized, which then causes the background to rise. Therefore, use bright field illumination, adjust the focus, and then acquire fluorescence images.

Handling and storage condition

Handling and storage condition
-20℃
Danger / harmful symbol mark

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ROS Assay Kit -Highly Sensitive DCFH-DA-

ROS Assay Kit -Highly Sensitive DCFH-DA-