Redefine the ROS Research

Reactive oxygen species (ROS) are mainly produced in mitochondria during ATP synthesis. ROS plays an essential role in signaling pathways and the immune system, but excess ROS is associated with diseases and cellular senescence. DCFH-DA is widely used for ROS detection, but it has some limitations, such as weak fluorescence signals in cells, leakage from cells after staining, and photo-oxidation phenomena during observation¹⁾. High data variability, high fluorescent background, and self-oxidation by observation light are the major challenges in ROS detection. Especially the self-oxidation may cause a false response, however Dojindo Laboratories' ROS Assay Kit -Photo-oxidation Resistant DCFH-DA- can solve these problems perfectly.

1) M. Afzal, et al.,"Method to overcome photoreaction, a serious drawback to the use of dichlorofluorescin in evaluation of reactive oxygen species" BBRC, 2003, 304, 619–624.

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

Resistant to Photo-oxidation

Unstimulated HeLa cells were stained with regular DCFH-DA or Photo-oxidation Resistant DCFH-DA and observed with excitation light every 5 minutes for fluorescence imaging.

Compatible with Immunostaining

After adding hydrogen peroxide, HeLa cells were stained with Photo-oxidation resistant DCFH-DA and mitochondrial marker Tom 20. As a result, the intracellular ROS level and mitochondrial morphology were clearly observed at the same time, which is very difficult for the existing ROS probe.

Super High-Sensitive ROS Detection

Comparison of Sensitivity: Microscope           

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.  Results indicated that the ROS Assay Kit-Highly Sensitive DCFH-DA was better at high-sensitivity detection of intracellular ROS than regular DCFH-DA in high-sensitivity detection of intracellular ROS was better than DCFH-DA.

Ex: 450-490 nm, Em: 500-550 nm (GFP filter), Scale bar: 50 μm

 Comparison of Sensitivity: Plate reader

In Lipopolysaccharide (LPS)-treated RAW 264.7 cells, after being stained with regular DCFH-DA, Highly Sensitive DCFH-DA, or Photo-oxidation Resistant DCFH-DA, the intracellular ROS levels were compared. The results showed that the Dojindo Laboratories’ probes could detect intracellular ROS with higher sensitivity.

Ex: 490 – 520 nm, Em:  510 – 540 nm

MitoBright ROS Deep Red has higher superoxide selectivity than Company T's existing product Red.

In addition, MitoBright ROS Deep Red has a special fluorescence spectrum (λex: 540 nm, λem: 670 nm), making it possible to co-staining with mitochondrial membrane potential reagent (JC-1, code: MT09, TMRE, MT-1, code: MT13), which is difficult for others mitochondrial superoxide reagent.

Company T's product Red tends to localize to DNA in cells, but MitoBright ROS Deep Red does not localize to DNA. This is because MitoBright ROS does not have the ability to bind to DNA. This avoids false positives due to binding to DNA and allows for more accurate observation of mitochondrial superoxide. 

HeLa cells were washed with HBSS and co-stained with MitoBright ROS Deep Red - Mitochondrial Superoxide Detection and ROS Assay Kit -Highly Sensitive DCFH-DA- (Total ROS detection), and separately treated with mitochondrial superoxide inducer Antimycin or hydrogen peroxide. As a result, intracellular total ROS and mitochondrial superoxide were observed separately.

General Protocol

Treated with H₂O₂

Treated with Antimycin

＜Imaging Conditions＞(Confocal microscopy)
Intracellular ROS: Ex = 488, Em = 490-520 nm
MitoBright ROS: Ex = 633 nm, Em = 640-700 nm
Scale bar: 10 μm

In ROS Assay Kit -Photo-oxidation Resistant DCFH-DA- stained RAW264.7 cells, after LPS (Lipopolysaccharide)-treated, the intracellular ROS level was simultaneously observed with a fluorescence microscope. The fluorescence intensity graph showed that, after 10 hours of LPS-treated, the intracellular ROS level was significantly increased.

Video

Intracellular ROS（Photo-oxidation Resistant DCFH-DA Dye）： GFP Filter (Ex = 450 - 490 nm, Em = 500 - 550 nm)

Cystine/glutamate antiporter (xCT) is frequently overexpressed in human cancers and promotes cystine uptake and glutathione biosynthesis*. This results in protection from oxidative stress and ferroptosis, a type of programmed cell death dependent on iron and characterized by the accumulation of lipid peroxides. We, therefore, treated A549 cells with erastin to inhibit xCT before evaluating intracellular uptake and redox balance

* Pranavi Koppula, et. al., Cancer Commun., 38:12 (2018)

As a result, we were able to get three conclusions:
・To compensate for the loss of cystine, the uptake of amino acids increased.
・The decrease in glucose uptake suggests that metabolic reprogramming may have taken place.
・The decrease in glutathione would have increased Fe²⁺, ROS, and Lipid peroxides – all hallmarks of Ferroptosis.

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