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.
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
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 (1×10
4 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% CO
2.
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 Condition>
Ex. 488 nm / Em. 500 – 560 nm
Scale bar: 50 μm
RAW264.7 cells
1. RAW264.7 (3×10
4 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% CO
2.
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 Properties