Commercially available enzyme-linked immunosorbant assay (ELISA) kits were used to measure the concentrations or activity of HMGB1 (Shino Test Corporation, ST51011), amylase (BioVision, K711), MPO (BioVision, K744), iron (Abcam, ab83366), MDA (Abcam, ab118970), and 8-OHdG (Cell Biolabs, STA-320) in indicated samples according to the manufacturer’s instructions. Data were normalized to protein or DNA concentration. In addition, C11-BODIPY probe (Thermo Fisher Scientific, D3861) was used to detect lipid ROS in cells.
C11 bodipy probe
Manufactured by Thermo Fisher Scientific
Sourced in United States
The C11-BODIPY probe is a fluorescent lipid peroxidation indicator. It is used to detect and quantify lipid peroxidation in biological samples.
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5 protocols using c11 bodipy probe
1
Quantifying Biomarkers in Biological Samples
2
Lipid Oxidation Assay
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Cells were cultured and treated with the indicated reagents. At the end of the treatment, cells were harvested and resuspended in 1 mL of DMEM medium supplemented with 10% BCS and a 10 µm C11 BODIPY probe (Thermo Fisher Scientific; cat #D3861). Next, the samples were incubated for 30 min at 37 °C with 5% CO2 in the dark and then washed with PBS. Samples were analyzed using a flow cytometer (Fortessa, BD Biosciences) with a fluorescein isothiocyanate (FITC) green channel and a Texas red channel.
3
Lipid Peroxidation Assays in RAW264.7 Macrophages
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RAW264.7 macrophages were seeded in 96-well plates (3 × 103 cells/well) and treated with several experimental treatments 24 h after cell adhesion. After stimulation, a lipid peroxide test kit (A106-1, Jiancheng, Nanjing, China) was used to evaluate the amount of lipid peroxide in each group. A microplate reader (Multiskan FC, Waltham, MA, USA) was used to measure the absorbance at 586 nm.
RAW264.7 macrophages were seeded in 6-well plates (1 × 106 cells/well) and treated with several experimental treatments 24 h after cell adhesion. Lipid peroxidation levels were detected using a C11BODIPY probe (D3861, Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated with 2 μM C11BODIPY for 30 min at 37 °C, then washed three times with PBS. Finally, cells were imaged using a fluorescence microscope (THUNDER DMi8, LEICA, Munich, Germany). The mean fluorescence intensity of C11BODIPY Green, which measures the amount of lipid ROS in the cell, was calculated using ImageJ software (NIH, Bethesda, MD, USA).
RAW264.7 macrophages were seeded in 6-well plates (1 × 106 cells/well) and treated with several experimental treatments 24 h after cell adhesion. Lipid peroxidation levels were detected using a C11BODIPY probe (D3861, Thermo Fisher Scientific, Waltham, MA, USA). Cells were incubated with 2 μM C11BODIPY for 30 min at 37 °C, then washed three times with PBS. Finally, cells were imaged using a fluorescence microscope (THUNDER DMi8, LEICA, Munich, Germany). The mean fluorescence intensity of C11BODIPY Green, which measures the amount of lipid ROS in the cell, was calculated using ImageJ software (NIH, Bethesda, MD, USA).
4
Measuring Oxidative Stress in HBE Cells
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HBE cells were sonicated with cell lysis buffer (Beyotime) and then centrifuged at 12,000 ×g for 10 min at 4 °C. The supernatant was collected for the MDA assay (Beyotime) following the manufacturer’s protocol. MDA levels were detected at a wavelength of 532 nm.
For the C11BODIPY probe (Invitrogen, USA), HBE cells were incubated with 1 μM C11BODIPY at 37 °C for 30 min in DMEM and then washed with PBS 3 times. The red and green fluorescence signals in the cells were viewed under a fluorescence microscope.
For the C11BODIPY probe (Invitrogen, USA), HBE cells were incubated with 1 μM C11BODIPY at 37 °C for 30 min in DMEM and then washed with PBS 3 times. The red and green fluorescence signals in the cells were viewed under a fluorescence microscope.
5
Quantifying Oxidative Stress and Iron Levels in Osteocytes
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To prevent interference from Dmp1-GFP fluorescence in IDG-SW3 cells after mineralization induction, we used unmineralized IDG-SW3 cells for fluorescence staining. A C11-BODIPY probe (D3861, Invitrogen, USA) was used to evaluate cellular lipid peroxidation. After the indicated treatments, osteocytes were incubated with C11-BODIPY working solution (2.5 μmol·L− 1) for 30 min prior to analysis. A FerroOrange probe (F374, Dojindo, Shanghai, China) was used to detect intracellular Fe2+. After the indicated treatments, osteocytes were washed with PBS solution and treated with FerroOrange working solution (1 μmol·L− 1) for 30 min. Finally, stained cells were observed using confocal scanning microscopy.
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