Lipid peroxide was analyzed by flow cytometry. Cell lines were plated in quadruplicate at the cell numbers indicated previously for the 12-well plate format. Cells were seed overnight and were subjected to various compound treatments for indicated times. Cells were then incubated for 30 minutes in live-cell imaging solution containing the pertinent ROS dye at the following concentrations: C-11 BODIPY (Invitrogen) (2 μM). Cells were then washed with PBS, trypsinized with 0.25% trypsin, and neutralized with 10% FBS in PBS. A minimum of 10,000 cells were analyzed per condition. For C-11 BODIPY, signal was analyzed in the FITC channel. Software analysis and histogram generation was carried out using FlowJo v10. For Lipid peroxidation confocal imaging, Cells were seeded at a density of 2.5×105 per well on coverslips placed in a 6-well dish and grown overnight in DMEM. Cells were treated with cystine-free medium for 16 hours, the coverslips were then mounted to a Teflon chamber. C11-BODIPY(Invitrogen) (5 μM) were added to each well 15 min before measurements. Reduced and oxidized C11-BODIPY were detected at emission 590 nm and 510 nm, respectively.
C11 bodipy
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, China
C11-BODIPY is a fluorescent lipid probe designed for visualization and quantification of lipid dynamics in live cells. It consists of a BODIPY fluorescent dye attached to an eleven-carbon alkyl chain. This probe can be used to monitor lipid-related cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Facscanto 2, by BD (9 mentions)
H2dcfda, by Thermo Fisher Scientific (9 mentions)
Mitosox, by Thermo Fisher Scientific (8 mentions)
Sytox green, by Thermo Fisher Scientific (7 mentions)
C11 bodipy, by BD (7 mentions)
Dcfh da, by Beyotime (5 mentions)
Bodipy 581 591 c11, by Thermo Fisher Scientific (5 mentions)
Propidium iodide, by Thermo Fisher Scientific (5 mentions)
Accuri c6, by BD (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Celltiter glo luminescent cell viability assay, by Promega (3 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (3 mentions)
Iron assay kit, by Abcam (3 mentions)
Erastin, by Merck Group (3 mentions)
Ferrostatin 1, by Merck Group (3 mentions)
Lipid peroxidation mda assay kit, by Beyotime (3 mentions)
Facscanto 2 flow cytometer, by BD (3 mentions)
Cumene hydroperoxide, by Merck Group (3 mentions)
C11 bodipy, by Beckman Coulter (3 mentions)
Cytoflex, by Beckman Coulter (3 mentions)
190 protocols using c11 bodipy
1
Lipid Peroxidation Analysis by Flow Cytometry
2
Quantifying Lipid ROS and Nitric Oxide
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After treatment with MC‐3T3 as prescribed, 50‐μM C11‐BODIPY (#D3861; Invitrogen, CA, USA) was added and incubated for 1 h. Excess C11‐BODIPY(boron‐dipyrromethene) was removed by washing the cells twice with PBS. After trypsinization, the labelled cells were resuspended in PBS and 5% foetal bovine serum. The oxidation of C11‐ BODIPY caused a shift in the fluorescence emission peak from 590 to 510 nm, which was proportional to lipid ROS generation, and was then analysed with a flow cytometer. To measure nitric oxide (NO) production in mouse hearts and MC‐3T3, the total nitric oxide assay kit (#S0021S, Beyotime, Shanghai, China) was used to quantify its stable metabolite (nitrite). A full‐wavelength multifunctional microplate reader JS‐THERMO Varioskan Flash (Thermo Fisher Scientific, USA) was employed to measure the absorbance.
3
Multimodal ROS Detection Assay
4
Measurement of Intracellular Lipid ROS
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Cells were incubated with DCFH-DA (10 μmol/L, Beyotime, Shanghai, China) at 37 °C for 30 min. Cells were then subjected to flow cytometry analysis for ROS detection. To detect intracellular lipid ROS, cells were incubated with C11-BODIPY (50 μmol/L, Thermo Fisher Scientific, Inc., MA, USA) at 37 °C for 1 h. Excess C11-BODIPY was removed by washing cells twice with PBS. Lipid ROS is proportional to the oxidation of polyunsaturated butadienyl portion of C11-BODIPY. When cells are oxidized, the released lipid ROS quench the originally red fluorescence and elicit green fluorescence under confocal microscope.
5
ROS Measurement in Endothelial Cells
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For total ROS measurement, MAECs with indicated treatments were added with 10 μmol/L DCFH-DA (Solarbio) and incubated at 37 °C for 20 min. For lipid ROS detection, MAECs from different groups were stained with 50 μM C11-BODIPY (Thermo Fisher) for 1 h. Redundant DCFH-DA or C11-BODIPY was eliminated by rinsing with PBS for three times. The labeled MAECs were analyzed on a microplate reader.
6
Oxidative Stress Measurement Protocols
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Cells were treated with different concentrations of test compound or drug. After 24 h, cells were incubated at a final concentration of 10 μM 2′,7′-DCFH-DA for 30 min at 37°C, after which they were washed, fixed with 4% paraformaldehyde for 15 min, and permeabilized (using 0.2% Triton X-100 in PBS) for 15 min under room temperature, dyed with DAPI, and immediately analyzed for fluorescence intensity under Leica Confocal Microscope (TCS SP8, Germany) with a ×63 objective lens.
If analyzed by flow cytometry, treated cells were washed 3 times with PBS, harvested, and suspended in PBS followed by flow cytometric analysis (Ex: 488 nm/Em: 510–555 nm).
If analyzed cells with BODIPY-C11 fluorescent dye, 1 × 106 cells were incubated with 5 μM BODIPY-C11 (Thermo Fisher Scientific) for 30 min at 37°C. Cells were washed 3 times with PBS, harvested, and suspended in serum-free medium followed by flow cytometric analysis (Ex: 488 nm/Em: 510–555 nm).
If analyzed by flow cytometry, treated cells were washed 3 times with PBS, harvested, and suspended in PBS followed by flow cytometric analysis (Ex: 488 nm/Em: 510–555 nm).
If analyzed cells with BODIPY-C11 fluorescent dye, 1 × 106 cells were incubated with 5 μM BODIPY-C11 (Thermo Fisher Scientific) for 30 min at 37°C. Cells were washed 3 times with PBS, harvested, and suspended in serum-free medium followed by flow cytometric analysis (Ex: 488 nm/Em: 510–555 nm).
7
Topoisomerase Inhibitors and Oxidative Stress
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B16-F1 and B16-F10 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). Topoisomerase inhibitors were purchased from Sigma-Aldrich (St. Louis, MO, USA; camptothecin, merbarone, teniposide, etoposide, XK469, ICRF-193) and Cayman Chemical (Ann Arbor, MI, USA; doxorubicin, moxifloxacin). NAC, LPA and HYD HCl were from Sigma-Aldrich. Fluorescent probes were from Life Technologies (Waltham, MA, USA; DDAOG, C11-BODIPY, CellROX Green, CellTrace Violet), eBioscience (San Diego, CA, USA; CV450) and Sigma-Aldrich (DAPI). Bafilomycin A1 was from Research Products International (Mount Prospect, IL, USA). Primary antibodies were from Millipore (Billerica, MA, USA; anti-γH2AX, clone JBW301) or Abcam (Cambridge, UK; anti-4-HNE, anti-OHdG). Fluorescent secondary antibodies were obtained from Thermo Pierce (Waltham, MA, USA). Cell culture reagents were from Life Technologies (DMEM, pen-strep solution, trypsin-EDTA) and Gemini Biosciences (West Sacramento, CA, USA; fetal bovine serum, stabilized l -glutamine).
8
Topoisomerase Inhibitors and Oxidative Stress
9
Ferroptosis Induction and Measurement
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In total, 150,000 cells per well were seeded on 6-well dishes one day prior to the experiment. On the next day, cells were treated with 300 nM of RSL3 or 3 µM of iFSP1 to induce ferroptosis. Cells were incubated with C11-BODIPY (581/591) (1 μM) for 30 min at 37 °C before they were harvested by trypsinisation. Subsequently, cells were resuspended in 500 μL of fresh PBS (DPBS, Gibco) and analyzed using a 488-nm laser excitation laser and fluorescence recorded on a (FACS Canto II, BD Biosciences). Data was collected from the FL1 detector (C11-BODIPY, Invitrogen) with a 502LP and 530/30 BP filter. At least 20,000 events were analyzed per sample. Data was analyzed using FlowJo Software version 10.8.1.
10
Quantifying Oxidative Stress in Cells
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The assessment of cytosolic and mitochondrial lipid peroxidation allows for the quantitative analysis of oxidative stress in the cells. HT22 cells were treated as indicated and stained after 16 h of incubation with 0.5 µg/mL/well C11-BODIPY (Invitrogen, Karlsruhe, Germany) or 1 µg/mL MitoPerOx (Abcam, Cambridge, UK; GB) for 30 min in the cell culture incubator. Afterward, cells were washed to remove the unbound probe and collected in a microcentrifuge tube. Filters for signal detection were adjusted to 525/30 nm for excitation of the green fluorescence signal and to 690/50 nm for emission of the red fluorescence signal. BODIPY and MitoPerOx oxidation were measured from at least 5.000 cells per replicate per condition and analyzed by recording the shift from red to green fluorescence with the Guava EasyCyte flow cytometer (Merck Millipore, Darmstadt, Germany).
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