The reactive oxygen species (ROS) level was analyzed using ROS Assay Kit -Photo-oxidation Resistant DCFH-DA- (Dojindo, kumamoto, Japan). Briefly, strains, pre-incubated in 5 mL of YPD at 30 °C overnight, were inoculated into 5 mL of fresh YPD at 1.0 OD600 and incubated at 30 °C for 4 h in test tubes with shaking at 200 rpm. Then, 1 mL of each culture was centrifuged at 5,000 × g for 2 min, and the supernatant was removed. The cells were treated according to the manufacturer’s protocol and the fluorescence was measured with λex = 500 nm and λem = 540 nm using a plate-reader (INFINITE M NANO + ; Tercan, Männedorf, Switzerland).
Ros assay kit photo oxidation resistant dcfh da
Manufactured by Dojindo Laboratories
Sourced in Japan
The ROS Assay Kit - Photo-oxidation Resistant DCFH-DA - is a laboratory product developed by Dojindo Laboratories. It is designed to detect and measure reactive oxygen species (ROS) in biological samples. The kit utilizes a photo-oxidation resistant DCFH-DA compound, which is a fluorescent probe that can be used to quantify ROS levels.
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Lab products found in correlation
Tcs sp8, by Leica (1 mentions)
Secondary hrp conjugated goat anti rabbit igg, by ABclonal (1 mentions)
Ferroorange, by Dojindo Laboratories (1 mentions)
Lipid peroxidation probe bdp 581 591 c11, by Dojindo Laboratories (1 mentions)
Erastin, by Selleck Chemicals (1 mentions)
Zen 3.2 blue edition software, by Zeiss (1 mentions)
Axio imager m2, by Zeiss (1 mentions)
Hoechst 33342, by Dojindo Laboratories (1 mentions)
4 protocols using ros assay kit photo oxidation resistant dcfh da
1
Quantifying Cellular Reactive Oxygen Levels
2
Reactive Oxygen Species Imaging
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The cells were stained for 30 min with DCFH‐DA dye in a 5% CO2 incubator at 37°C using the ROS Assay Kit (Photo‐oxidation Resistant DCFH‐DA, R253, DOJINDO) by following the manufacturer's instructions. The nucleus was stained with Hoechst 33,342 for cell localization. The images were captured using a confocal fluorescence microscope (TCS SP8, Leica), and the fluorescence intensity was calculated using ImageJ (Version: 2.0.0‐rc‐69/1.52p, http://imagej.net/Contributors ).
3
Lipid Peroxidation and ROS Assays
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FerroOrange (#F374), ROS Assay Kit -Photo-oxidation Resistant DCFH-DA (#R253) and Lipid Peroxidation Probe-BDP 581/591 C11 (#L267) were purchased from DOJINDO LABORATORIES (Kumamoto, Japan). Antibodies against GPX4 (#A1933), NCOA4 (#A5695), ACTB (#AC026), FTH1 (#A19544), LC3 (#A17424), KRAS (#A12704) as well as secondary HRP-conjugated Goat Anti-Rabbit IgG (#AS014), secondary HRP-conjugated Goat Anti-Mouse IgG (#AS003) were from ABclonal (Wuhan, China). Antibodies against CH25H (#ER1919-26) were from HUABIO (Hangzhou, China). Erastin (#S7242) was purchased from Selleckchem (Shanghai, China). Chloroquine (#HY-17589A) and wortmannin (#HY-10197) were purchased from MCE (Shanghai, China). 25-HC (#H1015) was purchased from Sigma-Aldrich (MO, USA).
4
ROS Measurement in Cells
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Intracellular ROS levels were assessed using fluorogenic probes with the ROS Assay Kit—Photo-oxidation Resistant DCFH-DA—(R253; Dojindo) following the manufacturer’s protocol. The cells were incubated for 30 min at 37 °C with the ROS Assay working solution and then treated with H2O2. After washing twice with DMEM, the cells were fixed with 4% paraformaldehyde and Hoechst 33342 (Dojindo) was added to detect the nuclei. The stained cells were observed by fluorescence microscopy (Axio Imager M2; Carl ZEISS AG, Oberkochen, Germany) at 550/605nm (excitation/emission). The fluorescence intensity derived from DCFH-DA was calculated as the average intensity value of the stained cells by ZEISS ZEN 3.2 (blue edition) software (Carl ZEISS AG).
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