As per the manufacturer’s protocol, lipid peroxidase levels were evaluated using Liperfluo reagent (Dojindo, Kumamoto, Japan). Cancer cells were seeded in 12-well plates and treated for 16 h. After the treatment period, cells were incubated with 10 μM Liperfluo for thirty minutes at 37 °C. After that, cells were gently washed twice with PBS, and fluorescence images were obtained using a confocal microscope.
Liperfluo reagent
Manufactured by Dojindo Laboratories
Sourced in Japan
Liperfluo is a reagent developed by Dojindo Laboratories. It is designed to measure lipid peroxidation levels in biological samples.
Automatically generated - may contain errors
3 protocols using liperfluo reagent
1
Lipid Peroxidation Evaluation in Cancer Cells
2
Quantifying Lipid Peroxidation in Cells
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To quantify lipid peroxidation, 15,000 cells per well were plated in 24-well plates and cultured for 24 h prior to exposure to CaOx (1 mM) for an additional 24 h. Lipid peroxidation was measured utilizing the Liperfluo reagent (Dojindo Laboratories, L248). Treated cells were incubated with 10 μM Liperfluo for 1 h at 37 °C to stain them. The fluorescence signal was subsequently quantified using a flow cytometer (Beckman Coulter, CytoFlex, USA), with data analysis performed via FlowJo 10.6.2 software. Additionally, the Lipid Peroxidation Probe -BDP 581/591 C11 assay (Dojindo Laboratories, L267) was employed for further lipid peroxidation assessment. Visualization of lipid peroxidation was achieved through an inverted fluorescence microscope (Olympus, IX71, Japan).
3
Fluorescent Probes for ROS Quantification
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Production of hydroxyl radical ( Á OH) and hypochlorous acid (HClO) were measured using the OxiORANGE reagent (Goryo Chemical, Sapporo, Japan). H 2 O 2 and NO were detected by HYDROP and diaminofluorescein-FM diacetate (DAF-FM DA) (Goryo Chemical), respectively. Cells plated in a 96-well Black IsoPlate (PerkinElmer, Waltham, MA, USA) were incubated with 0.5 µM OxiORANGE, 1 µM HYDROP, or 1 µM DAF-FM DA diluted in EMEM containing FBS for 30 min at 37 °C, and then washed with 100 µL Hank's balanced salt solution (HBSS) once. Fluorescence (Ex/Em = 535/595 nm for OxiORANGE, 485/ 535 nm for HYDROP and DAF-FM DA) was measured by using FilterMax F5 (Molecular Devices, San Jose, CA, USA). Normalized values were obtained by dividing the fluorescent intensities of HYDROP by protein concentration, those of DAF-FM DA by nuclear signal, and those of Oxi-ORANGE by nuclei number determined by Hoechst 33342 staining (Dojindo). Lipid peroxide was measured using the Liperfluo reagent (Dojindo). Cells were stained with 1 µM Liperfluo reagent diluted in EMEM without FBS for 30 min at 37 °C, and then observed under an all-in-one fluorescence microscope BZ-9000 (Keyence, Osaka, Japan).
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