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.

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. 

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.

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.

Sulfasalazine Alters Intracellular Metabolites and Increases ROS in A549 Cells

	After addition of sulfasalazine (SSZ), a known inhibitor of cystine/glutamate transporter (xCT), to A549 cells, we observed changes in intracellular glutathione (GSH), ATP, α-ketoglutarate (α-KG), ROS and glutamate release. The results indicated that the addition of SSZ The results showed that the addition of SSZ decreased intracellular ATP, glutathione (GSH) and glutamate release, and increased intracellular α-ketoglutarate and ROS. 　Cells: A549 cells (1 x 106 cells) Exposure time: 48 hours Product in Use - ATP Assay Kit-Luminescence - α-Ketoglutarate Assay Kit-Fluorometric - ROS Assay Kit -Highly Sensitive DCFH-DA-  - Glutamate release: Glutamate Assay Kit-WST - GSSG/GSH quantification Kit    Reference Shogo Okazaki et al., "Glutaminolysis-related genes determine sensitivity to xCT-targeted therapy in head and neck squamous cell carcinoma". Cancer Sci., 2019, doi:10.1111/cas.14182.

Induction of Ferroptosis by Erastin

Erastin is a known inducer of ferroptosis. By inhibiting the cystine transporter (xCT), erastin inhibits the uptake of cystine. Cystine is the raw material for GSH. Therefore, Erastin ultimately decreases the amount of GSH. Decreased GSH then results in lipid peroxide accumulation and induction of ferroptosis.
The following experimental examples show changes in each aforementioned index as a consequence of erastin stimulation. Measurements are made using Dojindo reagents.

Using erastin-treated A549 cells, we measured intracellular Fe²⁺, ROS, lipid peroxide, glutathione, glutamate release into the extracellular space, and cystine uptake. As a result, inhibition of xCT by elastin was observed and also the release of glutamate and uptake of cystine were decreased. Furthermore, elastin treatment decreased intracellular glutathione while it increased intracellular Fe²⁺ , ROS, and lipid peroxides.

Cell Line: A549
Incubation Conditions: 100 μmol/l Erastin/MEM, 37℃, 3h

Fluorescence Property