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.

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Comparison the Highly Sensitive DCFH-DA with DCFH-DA

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.

”The 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
Note: Comparisons based on observation conditions of Highly Sensitive DCFH-DA.

<Detection Condition>
Ex: 450-490 nm, Em: 500-550 nm（GFP filter）
Scale bar: 50 μm
HeLa cells


② Detection using microplate reader

<Detection Condition>
Ex. 490 – 520 nm / Em. 510 – 540 nm
HeLa cells


③ Detection using flow cytometer

<Detection Condition>
FITC laser gain: 215 V
HeLa cells

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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.

<Detection Condition>
Ex. 488 nm / Em. 500 – 560 nm
Scale bar: 50 μm
A549 cells


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^oC 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.

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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.

<Detection Condition>
Ex. 488 nm / Em. 500 – 560 nm
Scale bar: 50 μm
RAW264.7 cells


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^oC 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.

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Fluorescence Properties

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.

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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.

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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.

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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.

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. Tmoi 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.
7)	Cell (MDSCs Cell)	Flow Cytometer	Z. Xie, T. Kawasaki, H. Zhou, D. Okuzaki, N. Okada and M. Tachbana, "Targeting GGT1 Eliminates the Tumor-Promoting Effect and Enhanced Immunosuppressive Function of Myeloid-Derived Suppressor Cells Caused by G-CSF", Front. Pharmacol., 2022, doi:10.3389/fphar.2022.873792.
8)	Cell (TIG-1 Cell)	Fluorescent Microscope	Y. Fujita, M. Iketani, M. Ito, I. Ohsawa, "Temporal changes in mitochondrial function and reactive oxygen species generation during the development of replicative senescence in human fibroblasts", 2022, doi:10.1016/j.exger.2022.111866.