The fluorescent probe BODIPY-C11 (665/676) (Invitrogen, Waltham, MA, USA) was used to detect lipid peroxides by flow cytometry following a previously described [75 (link)]. Briefly, cells were treated with 500 nM BODIPY-C11 (665/676) for 30 min at 37°C. After incubation, cells were washing twice with PBS, trypsinized, and analyzed for fluorescence of the oxidized form of BODIPY-C11 using BD FACSCalibur, CellQuest Pro (BD). Flowjo V10.8.0 software was used for data analysis.
C11 bodipy
Manufactured by BD
Sourced in United States
C11-BODIPY is a fluorescent dye compound used in various laboratory applications. It functions as a lipid probe, enabling the visualization and analysis of lipid dynamics within biological systems.
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Lab products found in correlation
C11 bodipy, by Thermo Fisher Scientific (7 mentions)
Bodipy 581 591 c11, by Thermo Fisher Scientific (2 mentions)
Facscalibur, by BD (1 mentions)
Cellquest pro, by BD (1 mentions)
Facsdiva v8, by BD (1 mentions)
Lsm 880 microscope, by Zeiss (1 mentions)
Synergy neo multi mode plate reader, by Agilent Technologies (1 mentions)
Lsrii fortessa flow cytometer, by BD (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Accuri c6 plus, by BD (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Dhr123, by BD (1 mentions)
Dhr123, by Merck Group (1 mentions)
Lsr 2 flow cytometer, by BD (1 mentions)
Accuri c6, by BD (1 mentions)
Lipid peroxidation assay kit, by Abcam (1 mentions)
C11 bodipy dye, by Thermo Fisher Scientific (1 mentions)
Facscanto 2, by BD (1 mentions)
C11 bodipy, by ABclonal (1 mentions)
11 protocols using c11 bodipy
1
Lipid Peroxidation Detection by Flow Cytometry
2
Measuring Cellular Oxidative Stress Levels
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Cellular ROS levels were measured using an ROS detection kit (BioVision, Waltham, MA; #K936-100), as previously described48 (link): 50,000 cells were plated in wells and collected, and fluorescence intensity at Ex/Em = 495/529 nm was quantified using a BioTek Synergy Neo Multi-Mode Plate Reader (BioTek, Santa Clara, CA). A representative plot of three biological replicates is shown.
For lipid Peroxidation determination, cells were seeded in DMEM and washed twice in HBSS and treated with Oxaliplatin (Normal-dose, 5 µg ml−1; low-dose, 1 µg ml−1) in DMEM for the indicated time, then incubated in DMEM containing 2 mM BODIPY 581/591 C11 (Invitrogen, D3861) for 30 min at 37 °C. For imaging, slides were excited using the 488 and 565 nm laser and fluorescence measured from 505 to 550 nm and above 580 nm using Zeiss LSM880 microscope. Upon oxidation of the polyunsaturated butadienyl portion of the dye, there is a shift of the fluorescent emission peak from 590 nm to 510 nm, remaining lipophilic and thus reflecting lipid peroxidation. Further examination was performed through a BD laser analyzer using PE-Texas Red (PE-TR) filter (measuring non-oxidized BODIPY-C11) and fluorescein isothiocyanate (FITC) (measuring oxidized BODIPY-C11). A minimum of 10,000 cells were collected using BD FACS Diva v8.0 in each condition and analyzed using FlowJo v10.7 (Bioscience).
For lipid Peroxidation determination, cells were seeded in DMEM and washed twice in HBSS and treated with Oxaliplatin (Normal-dose, 5 µg ml−1; low-dose, 1 µg ml−1) in DMEM for the indicated time, then incubated in DMEM containing 2 mM BODIPY 581/591 C11 (Invitrogen, D3861) for 30 min at 37 °C. For imaging, slides were excited using the 488 and 565 nm laser and fluorescence measured from 505 to 550 nm and above 580 nm using Zeiss LSM880 microscope. Upon oxidation of the polyunsaturated butadienyl portion of the dye, there is a shift of the fluorescent emission peak from 590 nm to 510 nm, remaining lipophilic and thus reflecting lipid peroxidation. Further examination was performed through a BD laser analyzer using PE-Texas Red (PE-TR) filter (measuring non-oxidized BODIPY-C11) and fluorescein isothiocyanate (FITC) (measuring oxidized BODIPY-C11). A minimum of 10,000 cells were collected using BD FACS Diva v8.0 in each condition and analyzed using FlowJo v10.7 (Bioscience).
3
Lipid Peroxidation Quantification in Cell Lines
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Ramos, SUDHL4, SUDHL1, SR-786, and U937 cells (2.5 × 105 cells/ml) were treated with HDL NPs (50 nM) or PBS for 24, 48, or 72 h. Following treatment, C11-BODIPY (1 μM final concentration; Thermo Fisher Scientific) was added to each well and the cells were incubated for 30 min at 37 °C, 5% CO2. The cells were then washed twice with 1 X PBS, resuspended in ice-cold FACS buffer and C11-BODIPY fluorescence in the FITC channel quantified using the BD LSR II Fortessa flow cytometer. Data were analyzed using the FCS Express software.
4
Quantification of Lipid Peroxidation
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Cells were treated with CH for 2 h at 37 °C in a tissue culture incubator. Where indicated erastin (10 μm/ml) or ferrostatin-1 (2 μm/ml) was added at the same time as CH. BODIPY® 581/591 C11 (Thermo Fisher Scientific, 10 μm final concentration) was added during the last 30 min of culture. Cells were harvested by trypsinisation and immediately analysed by flow cytometry. C11 BODIPY was analysed by measuring fluorescence from both the green (FL-1) and red (FL-3) channels on a BD FACSVerse™ flow cytometer. To calculate the amount of lipid peroxidation, both the percentage gated (FL-1) was determined relative to controls, or, the mean fluorescence intensity (MFI) for both FL-1 and FL-3 channels were determined. The amount of lipid peroxidation was quantified by dividing the MFI(FL-1)/MFI(FL-3) from triplicate samples. In control cells the MFI(FL-3) is high resulting in a low FL-1:FL-3 ratio. However, following treatment with CH the MFI (FL-3) decreases whilst MFI (FL-1) increases resulting in a high FL-1:FL-3 ratio.
5
Quantifying Hepatic Lipid Peroxidation by MDA and Flow Cytometry
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To determine the extent of lipid peroxidation in the liver, hepatic malondialdehyde (MDA) contents were measured by using thiobarbituric acid (TBA) test. Briefly, after homogenization in ice-cold 0.01 M Tris-HCl (pH 7.4), hepatic tissues were centrifuged at 12,000× g for 15 min. After reaction with TBA, forming MDA-TBA adduct was estimated by measuring absorbance at 525 nm. MDA contents were normalized with total protein contents of liver homogenates and represented as nM MDA/mg protein.
To measure the lipid peroxidation in HepG2 cells, we conducted a flow cytometry using C11-BODIPY (Thermo Fisher Scientific, Rockford, IL, USA) dye. Cells were pretreated with MSH (100–1000 µg/mL, 1 h), and subsequently incubated with tBHP (200 µM) and C11-BODIPY for 3 h. After treatment, fluorescence intensity was measured at 485/530 nm (excitation/emission wavelengths) using an automated microplate reader (Infinite 200 PRO, Tecan, Männedorf, Switzerland). Additionally, the treated cells were detached by trypsinization, and the percentage of cells expressing high intensity of C11-BODIPY was analyzed using a flow cytometry (Accuri™C6 Plus; BD Biosciences, San Diego, CA, USA). A total of 10,000 events were recorded in each experiment.
To measure the lipid peroxidation in HepG2 cells, we conducted a flow cytometry using C11-BODIPY (Thermo Fisher Scientific, Rockford, IL, USA) dye. Cells were pretreated with MSH (100–1000 µg/mL, 1 h), and subsequently incubated with tBHP (200 µM) and C11-BODIPY for 3 h. After treatment, fluorescence intensity was measured at 485/530 nm (excitation/emission wavelengths) using an automated microplate reader (Infinite 200 PRO, Tecan, Männedorf, Switzerland). Additionally, the treated cells were detached by trypsinization, and the percentage of cells expressing high intensity of C11-BODIPY was analyzed using a flow cytometry (Accuri™C6 Plus; BD Biosciences, San Diego, CA, USA). A total of 10,000 events were recorded in each experiment.
6
Quantifying Lipid and Cytosolic ROS
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Lipid ROS and ROS measurements were performed as described before16 (link). Briefly, cells were seeded in 6 well plates and stimulated for ferroptosis, namely, 2 h stimulation with ML162 and RSL-3 and 8 h stimulation with Erastin. For iGPX4KD, lipid ROS and cytosolic ROS were measured in 1–2 h intervals for 8 h. Cells were collected, washed with PBS and resuspended in PBS in the presence of 2 μM C11-BODIPY (#D3861, Molecular Probes, USA) for lipid ROS measurement or 1 µM of DHR123 (#D-1054, Sigma Aldrich) for cytosolic ROS measurement. After a 10 min incubation at 37o C, cells were washed and the fluorescence intensity of oxidized C11-BODIPY or DHR123 was measured using BD LSR II flow cytometer in Fl-1 channel. The extent of cell death was measured by adding 0.5 µM DRAQ7 or 1.25 µM SytoxBlue®. Data were analyzed using FlowJo 10.2 software. Only non-permeabilized cells were used for the analysis.
7
Lipid ROS and Total GSH Measurement
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Cells were treated for 24 h with 6 μM RSL‐3 for the lipid ROS assay, after which they were trypsinized and resuspended in DMEM media with 10% FBS and 10 μM of C11‐BODIPY (D3861, Thermo Fisher). The samples were then incubated at 37°C and 5% CO2 for 30 min while being shielded from light. To get rid of extra C11‐BODIPY, PBS was used to wash the cells twice. Dr. Shuang Liu oversaw the use of a flow cytometer (Fortessa, BD Biosciences) with fluorescein isothiocyanate green channel and Texas red channel to measure the fluorescence of C11‐BODIPY581 = 591.
For the detection of Total GSH, after 24 h of incubation with 6 μM RSL‐3, cells were digested and collected in centrifuge tubes, and three volumes of ice‐cold 5% metaphosphoric acid were added immediately then frozen and thawed twice using liquid nitrogen and 37°C water. The supernatant was utilized for total GSH measurement in accordance with the protocol after 10 min of centrifugation at 12,000 rpm and 4°C.
For the detection of Total GSH, after 24 h of incubation with 6 μM RSL‐3, cells were digested and collected in centrifuge tubes, and three volumes of ice‐cold 5% metaphosphoric acid were added immediately then frozen and thawed twice using liquid nitrogen and 37°C water. The supernatant was utilized for total GSH measurement in accordance with the protocol after 10 min of centrifugation at 12,000 rpm and 4°C.
8
Lipid Peroxidation Measurement by C11-BODIPY
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A total of 10 µM C11-BODIPY (Thermo Fisher Scientific, Inc.) was added to the cell suspension (1x106 cells/ml), and the suspension was incubated in the dark at 37˚C for 30 min. Cells were then washed with PBS, and the fluorescence intensity of the dye C11-BODIPY was measured using an Accuri™ C6 flow cytometer (BD Biosciences, Inc.). Data were analyzed using FlowJo version 7.6.1 (FlowJo LLC).
9
Lipid Peroxidation and ROS Measurement
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Malonaldehyde (MDA) level was assayed with lipid peroxidation assay kit (BioVision). Cell lysate supernatant was harvested and incubated with 600 μL thiobarbituric acid reagent at 95°C for 60 min. Each reaction mixture was transferred 200 μL to a 96‐well plate, followed by MDA measurement.
Cells were dyed with C11‐BODIPY (Thermo Fisher) for 30 min following the manufacturer's specifications. Under the well without C11‐BODIPY as control, cells were washed and detected through flow cytometer (BD).
Cells were dyed with C11‐BODIPY (Thermo Fisher) for 30 min following the manufacturer's specifications. Under the well without C11‐BODIPY as control, cells were washed and detected through flow cytometer (BD).
10
Lipid ROS Quantification in Cancer Cells
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C11-BODIPY dye (10 µM; D3861; Thermo Fisher Scientific, Waltham, MA, USA) was used for lipid ROS staining according to the manufacturer’s instructions, with cumene hydroperoxide as a positive control. Briefly, stably transfected or parental 786-O and Caki-1 cancer cells were collected after treatment with URB597 and RSL3 singly or in combination (URB597 + RSL3) for 2 days and 10 µM C11-BODIPY-containing medium for 1 h. The lipid ROS level was determined by flow cytometry analysis (FACS Canto II; BD Biosciences).
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