FerroOrange

$140.00$320.00

High sensitivity for intracellular Fe2+ detection
Suitable for live cell imaging
Applicable for plate reader assay

Kit Contents: 1 tube 24μg x 1; 3 tubes 24μg x 3

Storage Condition: Store at 0-5oC and protect from light
Shipping Condition: Ambient Temperature

Ferroptosis Product Selection Guide

 

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DescriptionReferencesQ & AManualS.D.S

Product Description
Iron is the most abundant transition metal element within organisms, and it participates in various physiological activities. Recently, free iron in living cells has attracted attention because its high reactivity may be related to cellular damage or death. Free iron exists in its stable redox states, namely ferrous ion (Fe2+) and ferric ion (Fe3+)). In living cells, understanding the behavior of Fe2+) is considered more important than understanding that of Fe3+) because of the intracellular reductive environment, metal transporters, and the water solubility of Fe2+). In 2012, Ferroptosis was proposed as one of new cell deaths. Ferroptosis is studied as non apoptotic cell death caused by accumulation of iron ion-dependent lipid peroxide. FerroOrange is a novel fluorescent probe that enables live-cell fluorescent imaging of intracellular Fe2+, used in ferroptosis research.

Learn more >> Ferroptosis Detection


Ferrous ion (Fe2+) Detection

FerroOrange (F374) Mito-FerroGreen (M489)
Localization Intracellular Mitochondria
Fluorescent Property λex: 543 nm, λem: 580 nm λex: 505 nm, λem: 535 nm
Instrument (filter) Fluorescence microscope,
plate reader (Cy3)
Fluorescence microscope
(FITC, GFP)
Sample Live cell Live Cell
The number of assays 1 tube (24 µg)
17 assays at 35 mm dish
(final concentration 1 µmol/L)
1 set (50 µg x 2)
10 assays at 35 mm dish
(final concentration 5 µmol/L)

Experimental Example
HeLa cells treated with chelator of iron 2,2′-bipyridyl (Bpy) (100 μmol/L) or Ammonium iron (II) sulfate (100 μmol/L) were prepared. The change of intracellular Fe2+in HeLa cells was detected by the FerroOrange.

<Fluorescence Microscope>
Change of intracellular Fe2+in HeLa cells detected by FerroOrange

Ex/Em = 561 nm/570-620 nm, Scale bars 20 μm
Left Control
Middle Ammonium iron (II) sulfate and 2,2′-Bipyridyl (Bpy) treated
Right Ammonium iron (II) sulfate treated
The fluorescence intensity of FerroOrange was increased in HeLa cells treated with Ammonium iron (II) sulfate compared with the findings in untreated cells; conversely, its fluorescence intensity was decreased in cells treated with Bpy.

<Plate Reader Assay>
The change of intracellular Fe2+in HeLa cells quantified by FerroOrange
Ex/Em = 543 nm/ 580 nm
Left Control
Middle Ammonium iron (II) sulfate and 2,2′-Bipyridyl (Bpy) treated
Right Ammonium iron (II) sulfate treated
The change of intracellular Fe2+in HeLa cells was quantified by the FerroOrange.

<Metal Ion Selectivity>
Metal Ion Selectivity
Ex/Em = 543 nm/ 580 nm
2 μL of 1 mmol/L FerroOrange and 2 μL of 10 mmol/L from each metal were added to 1 mL of 50 mmol/L HEPES Buffer (pH7.4). The fluorescence intensity was measured after the reaction, for 1hr, at room temperature.

<Excitation and Emission Spectra>
Excitation and Emission Spectra


Applications

<High-throughput methods for monitoring subcellular labile Fe2+>

FerroOrange is ready to use for a 96-well-plate-based high-content imaging of labile Fe(II) in living cells. In the following article, Dr. Hirayama etc. were able to conduct high-throughput screening of a chemical library containing 3399 compounds.

High-Throughput Screening for the Discovery of Iron Homeostasis Modulators Using an Extremely Sensitive Fluorescent Probe

*FerroOrange is mentioned as “RhoNox-4” in the reference.
*FerroOrange was commercialized under the advisory of Dr. Hideko Nagasawa and Dr. Tasuku Hirayama (Gifu Pharmaceutical University).

 

<Ferrous ion (Fe2+) detection in mouse liver in ferroptosis research>

In the following article, ferrous ions (Fe2+) were detected in mouse livers using FerroOrange (Ferroptosis research).
*mice were fed on methionine- choline deficient diet
For details about the experiment, please visit the reference below.

Targeting Ferroptosis Alleviates Methionine-Choline Deficient (MCD)-diet Induced NASH by Suppressing Liver Lipotoxicity

 


Co-staining with Each Organelle Dye Reagent
FerroOrange was co-stained with each organelle’s dye reagents.
HeLa cells were stained with organelle’s dye and washed.
Then, FerroOrange was added to the cells and cells were observed under the fluorescent microscope.

 

Co-staining with ER Staining Dye
Co-staining with ER Staining Dye
<Detection Condition>
FerroOrange: Ex. 561 nm, Em. 570-620 nm
ER Tracker Green (ER Dye): Ex. 488 nm, Em. 510-555 nm
Scale bars: 10 µm

 

Co-staining with Mitochondrial staining Dye
Co-staining with Mitochondrial staining Dye
<Detection Condition>
FerroOrange: Ex. 561 nm, Em. 570-620 nm
MitoBright Deep Red (Mitochondrial Dye): Ex. 640 nm, Em. 650-700 nm
Scale bars: 10 µm

 

Co-staining with Golgi Complex Staining Dye
Co-staining with Golgi Complex Staining Dye
<Detection Condition>
FerroOrange: Ex. 561 nm, Em. 570-620 nm
BODIPY FL (Golgi Complex Staining Dye): Ex. 488 nm, Em. 510-555 nm
Scale bars: 10 µm

No. Sample Instruments Publications
1) Cell
(HeLa)
Fluorescence
microscope
K. Tomita, M. Fukumoto, K. Itoh, Y. Kuwahara, K. Igarashi, T. Nagasawa, M. Suzuki, A. Kurimasa and T. Sato, “MiR-7-5p is a key factor that controls radioresistance via intracellular Fe2+ content in clinically relevant radioresistant cells.”Biochem Biophys Res Commun.., 2019,doi: 10.1016/j.bbrc.2019.08.117.
2) Cell
(Endothelial cells)
Fluorescence
microscope
Y. Wang and M. Tang, “PM2.5 induces ferroptosis in human endothelial cells through iron overload and redox imbalance”Environ. Pollut.2019, 264, doi: 10.1016/j.envpol.2019.07.105.
3) Cell
(MCF7-ADR)
Fluorescence
microscope
S Guo, X Yao, Q Jiang, K Wang, Y Zhang, H. Peng, J. Tang and W. Yang , “Dihydroartemisinin-Loaded Magnetic Nanoparticles for Enhanced Chemodynamic Therapy”Front Pharmacol 2020, 11, 226.
4) Cell
(293T)
Fluorescence
microscope
R. A. Weber, F. S. Yen, S.P.V. Nicholson, H. Alwaaseem, E.C. Bayraktar,M. Alam, R. C. Timson, K. La, M. Abu-Remaileh, H. Molina and K. Birsoy, “Maintaining Iron Homeostasis Is the Key Role of Lysosomal Acidity for Cell Proliferation”Mol. Cell2020, 77, 1-11.
5) Cell
(SK-HEP-1)
Fluorescence
microscope
X. Li, T. Wang, X. Huang, Y. Li, T. Sun, S. Zang, K. Guan, Y. Xiong, J. Liu and H. Yuan , “Targeting ferroptosis alleviates methionine‐choline deficient (MCD)‐diet induced NASH by suppressing liver lipotoxicity”Liver Int., 2020,  doi:10.1111/liv.14428.
6) Cell
(HepG2)
Fluorescence
microscope
Microplate reader
Imaging cytometer
T. Hirayama, M. Niwa, S. Hirosawa and H. Nagasawa, “High-Throughput Screening for the Discovery of Iron Homeostasis Modulators Using an Extremely Sensitive Fluorescent Probe”ACS Sens., 2020,doi: 10.1021/acssensors.0c01445.
Note: FerroOrange is mentioned as “RhoNox-4” in the reference.
Are there any tips for successful assay?
1. Please do not change the media after adding the FerroOrange. By changing the media, the FerroOrange dye can leak out of cells.
2. For data reliability, we recommend preparing Bpy (2,2‘-bipyridine) or ammonium iron sulfate (II) treated cells as the control for comparison with FerroOrange data.
3. If the cell samples have difficulty in staining, please increase the concentration of FerroOrange working solution 1 μmol/L higher than the recommended concentration. We recommend 1-5μmol/L.
How can I use FerroOrange with a plate reader?
Please refer to the following experimental example:

<Sample>
A: No dye added (HeLa cells only)
B: Bpy (2,2‘-bipyridine) treated HeLa cells
C: Ammonium iron sulfate (II) treated HeLa cells

<Method>
1. Add 100μL HeLa cell suspension to each well of 96 well plate (black with clear bottom) making the final concentration 10,000 cells/well. Incubate overnight in the 5% CO2 at 37 ℃.
2. Wash Sample C with 100 μL MEM (no FBS) media three times
3. Add 100 μL ammonium iron sulfate (II)/MEM (no FBS) solution (final concentration: 100 μmol/L) to Sample C’s wells. Incubate for 30 minutes in the 5% CO2 at 37 ℃.
4. Wash cells with 100 μL HBSS three times
5. Add 100 μL of 1 μmol/L FerroOrange working solution to Sample A and Sample C’s wells. Add 100 μL of HBSS solution containing FerroOrange (final concentration: 1 μmol/L) and Bpy (final concentration: 100 μmol/L) to Sample B’s wells. Incubate for 30 minutes in the 5% CO2 at 37 ℃.
6. Measure the fluorescent intensity (Ex. 543 nm, Em. 580nm) using a fluorescence microplate reader.

What is the recommended filter?
Excitation: 530-565 nm; Emission: 570-620 nm

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Intracellular Fluorescent Probes

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