The intensely fluorescent BODIPY is an effective tracer of lipid trafficking (Bai et al. 1997 (link)). The C11-BODIPY 581/591 kit (D3861, Thermo Fisher) was used to test the LPO concentration in cells following the manufacturer’s instructions. Briefly, after cell treatment, 5 µM reagent was added to 96-well plates, and the cells were incubated for 30 min at 37 °C, washed with PBS three times, stained with 5 µg/ml Hoechst (33,342, Thermo Fisher) for 30 min at 37 °C, photographed under a fluorescence microscope (DM IL LED, Leica) and analyzed with Fiji software.
Bodipy 581 591 c11 kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BODIPY 581/591 C11 kit is a fluorescent probe designed for the detection and quantification of lipid peroxidation in biological samples. The kit utilizes a BODIPY-based dye that undergoes a shift in fluorescence emission upon oxidation, allowing for the monitoring of oxidative stress in cells and tissues.
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7 protocols using bodipy 581 591 c11 kit
1
Fluorescent BODIPY Lipid Trafficking
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Lipid ROS Evaluation by C11 BODIPY
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For the evaluation of lipid ROS generation in vitro, C11 staining by C11 BODIPY 581/591 kit (ThermoFisher, catalog # C10445) was used in our study. After the treatment, cell culture media was removed, and 10 μM Image-iT® Lipid Peroxidation Sensor (Component A) was applied to the cells for 30 min at 37 °C. PBS solution was then used to wash the cells three times, and the cells were immediately examined by fluorescence microscopy.
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Lipid ROS Evaluation via C11 BODIPY
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C11 staining with a C11 BODIPY 581/591 kit (Thermo Fisher, C10445, Waltham, MA, USA) was employed to evaluate lipid ROS generation in cells. We removed the cell culture media from each group after treatment and added 10 µM Image-iT® Lipid Peroxidation Sensor (Component A, Thermo Fisher, Waltham, MA, USA) to the cells and incubated the cells for 30 min at 37 °C, with nuclei stained using Hoechst dye. We washed the cells subsequently and evaluated lipid ROS generation via fluorescence microscopy.
4
Lipid Peroxidation Evaluation in GSCs
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The level of lipid peroxidation in GSCs was evaluated using the BODIPY 581/591 C11 kit (Thermo Fisher Scientific). Briefly, according to the manufacturer's instructions, GSCs were stained with 2 μM C11-BODIPY (581/591) probe. The oxidized BODIPY (O-BODIPY) and the reduced BODIPY (R-BODIPY) were visualized at excitation/emission wavelengths of 488/510 (traditional FITC filter set) and 581/591 nm (Texas Red filter set), respectively, utilizing a confocal microscope (LSM 880; Carl Zeiss AG, Jena, Germany). The relative fluorescence intensity of the C11-BODIPY probe was analyzed using Image J software (NIH, Bethesda, MD, USA).
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Lipid ROS Detection in Glioma Stem Cells
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The lipid ROS was detected using a BODIPY 581/591 C11 kit (Thermo Fisher Scientific, Waltham, MA, USA). The GSCs were seeded in 6-well plates at 5 × 104 cells/well for 24 h. Then GSCs were stained with 2 μM C11-BODIPY (581/ 591) probe according to the manufacturer’s instructions. GSCs were visualized using a laser scanning confocal microscope (Olympus) and analyzed by Image J software (NIH, Bethesda, MD, USA). The oxidized BODIPY (O-BODIPY) were observed at excitation/emission wavelengths of 488/ 510 (traditional FITC filter set), while the reduced BODIPY (R-BODIPY) were observed at excitation/emission wavelengths of 581/591 nm (Texas Red filter set).
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Lipid Peroxidation Measurement Using BODIPY-C11
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The BODIPY 581/591 C11 kit (D3861, Thermo Fisher Scientific) was used to measure lipid peroxidation levels. After treatment with IKE or CD for the indicated times, the cells were incubated with the BODIPY-C11 probe, which was added to medium at a final concentration of 5 µM for 30 min at 37 °C. Next, the cells were digested with trypsin, collected and centrifuged at 1000 rpm for 5 min. Then, the cells were resuspended in 300 µL PBS and transferred into tubes. Finally, cell fluorescence was acquired on a CytoFLEX-3 cytometer (Beckman Coulter). Oxidation of the dye causes the fluorescence emission peak to shift from 590 nm to 510 nm. The BODIPY-C11 value was calculated as the ratio of the oxidized probe to reduced probe fluorescence.
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Lipid ROS Quantification with BODIPY 581/591
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The lipid ROS levels were determined using the BODIPY 581/591 C11 kit (Thermo Fisher Scientific, Inc.). Cells were seeded into 6-well plates at a density of 4x104 cells/well and were then treated with 2 µM C11-BODIPY (581/591) probe, according to the manufacturer's instructions. The cells were visualized using a fluorescent inverted microscope. Image J software (version 1.42; National Institutes of Health) was used for analysis.
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