The relative intracellular levels of lipid peroxidation and Fe2+ were measured by the C11-BODIPY lipid probe (Invitrogen, Carlsbad, CA) and FerroOrange (Dojindo). The cells were gently rinsed with PBS three times, and the adherent cells were digested with trypsin. After digestion, centrifugation was conducted to collect cell pellets, which were resuspended with 100-µl living cell image solution (Thermo Fisher Scientific), and incubated with 0.1 µl C11-BODIPY (Invitrogen, 1:1000) and 0.2 µl FerroOrange (Dojindo, 1:500) in the dark for 30 min. Next, to blow and mix the cells, a 400-µl living cell image solution was added. The wavelength of FerroOrange excitation light was 543 nm, and the wavelength of emission light was 580 nm. Oxidation of the polyunsaturated butadienyl portion of the C11-BODIPY resulted in a shift of the fluorescence emission peak from ~ 590 to ~ 510 nm. The relative levels of lipid peroxidation and Fe2+ were measured by flow cytometry, which was quantified by the FITC/PE value and the PE value, respectively (Cheloni and Slaveykova 2013 ; Mei et al. 2020 (link)).
Ferroorange
Manufactured by Dojindo Laboratories
Sourced in Japan, China, United States
FerroOrange is a fluorescent iron indicator for the quantitative detection of ferrous (Fe²⁺) and ferric (Fe³⁺) iron in biological samples. It exhibits an increase in fluorescence upon binding to iron.
Automatically generated - may contain errors
Lab products found in correlation
Mito ferrogreen, by Dojindo Laboratories (14 mentions)
Liperfluo, by Dojindo Laboratories (10 mentions)
Bodipy 581 591 c11, by Thermo Fisher Scientific (10 mentions)
Dcfh da, by Beyotime (7 mentions)
Hoechst 33342, by Thermo Fisher Scientific (7 mentions)
Fluorescence microscope, by Olympus (4 mentions)
Erastin, by Selleck Chemicals (4 mentions)
Hanks balanced salt solution (hbss), by Thermo Fisher Scientific (4 mentions)
Lsm 780, by Zeiss (4 mentions)
Iron assay kit, by Merck Group (3 mentions)
Lsm 980, by Zeiss (3 mentions)
Dihydroethidium, by Beyotime (3 mentions)
Iron assay kit, by Abcam (3 mentions)
Acsl4, by ABclonal (3 mentions)
Ferroorange, by Thermo Fisher Scientific (3 mentions)
Biotek cytation 5, by Agilent Technologies (3 mentions)
Lipid peroxidation mda assay kit, by Beyotime (3 mentions)
Fv3000, by Olympus (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Propidium iodide, by Thermo Fisher Scientific (3 mentions)
164 protocols using ferroorange
1
Intracellular Lipid Peroxidation and Iron Assessment
2
Quantifying Intracellular Ferrous Iron
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To determine the intracellular ferrous iron content, FerroOrange (Dojindo, F374) was used according to the manufacturer's protocol. Fibroblasts were seeded in 96-well plates at 3 × 104 cells/well, cultured to 70–80% confluence, and washed twice with PBS. The cells were then treated with 1 μM FerroOrange and 1 μM Hoechst 33342 (DOJINDO, 346-07951) in HBSS (Wako, 085-09355) for 30 min at 37°C. The cells were fixed with 4% PFA/PBS for 15 min at 25°C. After washing with PBS, fluorescence intensities were captured using a multimode plate reader (EnVision, Perkin Elmer) and fluorescence images were captured using a fluorescence microscope (BZ-X810, Keyence).
3
Fluorescent Imaging of Fe2+ in Cells
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FerroOrange (Dojindo Laboratories, Japan, M489) is a novel fluorescent probe for fluorescence imaging of Fe2+ in living cells. Drug-treated cells were washed three times with HBSS, then 1 μmol/l FerroOrange working solution was added to the six-well plates. Finally, the six-well plates were incubated in a CO2 incubator for 30 minutes for imaging by fluorescence microscopy.
4
Intracellular Ferrous Ion Quantification
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Intracellular ferrous ions levels were assessed using FerroOrange (Dojindo, Japan) [33 (link)]. Cells were then washed with HBSS for 3 times, incubated with 1 μM FerroOrange for 30 min at 37 °C, 5% CO2 in an incubator. Finally, the fluorescence was detected by an inverted fluorescence microscope.
5
Intracellular Iron Quantification in Oocytes
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Intracellular Fe2+ levels were examined at the end of the IVM period. The oocytes in each group were thoroughly washed in prewarmed PBS-PVA medium and assessed using the fluorescent probe Ferro Orange (Dojindo, F374) for 30 min. Images of the fluorescence signals were captured as TIFF files using a digital camera connected to a fluorescence microscope. The same procedures were followed for all groups of oocytes, including incubation, rinsing, mounting, and imaging. The fluorescence signal intensities of the oocytes in each group were analyzed via National Institutes of Health (NIH) ImageJ software (NIH, Bethesda, MD, United States).
6
Retinal Iron Quantification and Localization
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Serum and retinal iron levels (total iron, ferrous iron, and ferric iron) were measured using an Iron Assay Kit (MAK025; Sigma) according to the manufacturer’s instructions. Perl’s staining was used to detect the ferric iron distribution in each layer of the retina. Briefly, deparaffinized and rehydrated retinal paraffin sections (3 µm) were stained using a Prussian blue staining kit (G1029; Servicebio) according to the recommended protocol. FerroOrange (F374; Dojindo, Beijing, China) was applied to reveal the distribution and expression of ferrous iron in the cytoplasm of living R28 cells. Briefly, the R28 cell culture medium was replaced with serum-free medium containing 1 μM FerroOrange, and then the R28 cells were incubated at 37 °C with an atmosphere containing 5% CO2 for 30 min before being detected using a confocal fluorescence microscope (Leica, Frankfurt, Germany).
7
Measuring Iron and ROS in Cells
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Assay kits were used to measure iron concentrations and ROS in cells. The cells were encased in black 96-well plates and stimulated with the drug for 24 h. FerroOrange (Dojindo Laboratories, Japan) dispersed in 1,640 was added to the cells and incubated at 37°C for 30 min. A fluorescence microplate reader was used to read at Ex: 543 nm and Em: 580 nm. For ROS, cells were incubated with DCFH-DA (Najing Jiancheng, catalog number: E004-1-1) at 37°C for 30 min. After 0.5 h of incubation, the remaining dye was washed off with PBS. The generated fluorescence intensity was measured with a fluorometer at Ex: 488 nm and Em: 525 nm.
8
Quantifying Intracellular Iron Levels
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The HT22 cells were seeded in 24-well plates at a density of 2.5 × 104 cells per well. After being treated with D-gal or D-gal and VD for 24 h, the cells were cultured with 1 μM FerroOrange(Dojindo, Kumamoto, Japan) at 37 °C for 30 min. Additionally, cell nuclei were stained with Hoechst 33342 (Beyotime Biotechnology, Shanghai, China) for 5 min at room temperature. The fluorescence was observed under a fluorescence microscope and captured in photographs.
9
Intracellular and Mitochondrial Iron Imaging
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FerroOrange and Mito-FerroGreen probes (DojinDo, Japan) were employed to assess intracellular and mitochondrial iron content, respectively. HCM and C2C12 cells were seeded into 24-well plates and exposed to atorvastatin at the concentrations of 40 uM alone, or other treatments for 24 h. After that, the cells were stained with FerroOrange (1 μM) or Mito-FerroGreen (5 μM) probes along with Hoechst 33,342 (5 μg/ml). After 30 min of lucifuge incubation, the cells were then evaluated by a Leica SP8 confocal fluorescence microscope (Leica Microsystems, Germany).
10
Ferrous Iron Staining and Imaging
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After treating the cells according to the grouping, the cells were washed two times using DMEM without FBS. Subsequently, Ferro Orange (1 μmol/L; λex: 543 nm, λem: 580 nm; Dojindo, Kumamoto, Japan) working solution was prepared using FBS-free DMEM according to the manufacturer’s instructions, incubated at 37 °C in a 5 % CO2 incubator for 30 min, and finally photographed by multifunctional microplate detection system (CYTATION5, BIOTEK, USA).
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