Product DescriptionIron 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

Experimental ExampleHeLa 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>

Ex/Em = 561 nm/570-620 nm, Scale bars 20 μmLeft ControlMiddle Ammonium iron (II) sulfate and 2,2′-Bipyridyl (Bpy) treatedRight Ammonium iron (II) sulfate treatedThe 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>Ex/Em = 543 nm/ 580 nmLeft ControlMiddle Ammonium iron (II) sulfate and 2,2′-Bipyridyl (Bpy) treatedRight Ammonium iron (II) sulfate treatedThe change of intracellular Fe²⁺in HeLa cells was quantified by the FerroOrange.

<Metal Ion Selectivity>Ex/Em = 543 nm/ 580 nm2 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.

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 dietFor 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 ReagentFerroOrange 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<Detection Condition>FerroOrange: Ex. 561 nm, Em. 570-620 nmER Tracker Green (ER Dye): Ex. 488 nm, Em. 510-555 nmScale bars: 10 µm

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

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

Excitation and Emission Spectra

Please refer to the following experimental example:

<Sample>A: No dye added (HeLa cells only)B: Bpy (2,2‘-bipyridine) 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 times3. 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

6. Measure the fluorescent intensity (Ex. 543 nm, Em. 580nm) using a fluorescence microplate reader.

<Usage Example>1. HeLa cells (1 x10⁵ cells/well) in MEM (10% fetal bovine serum, 1% penicillin-streptomycin) were seeded on a 6 well plate and were cultured at 37^oC in a 5% CO₂ incubator overnight.2. The cells were washed with serum-free medium (2 mL) three times. Then, serum-free medium (1 mL) was added to the cells.3. 10 mmol/L Ammonium iron (II) sulfate (10 μL) was added to wells (The final concentration: 100 μmol/L).4. To mix Ammonium iron(II) sulfate and serum-free medium, the entire medium was pipetted up from wells and then immediately pipetted back one time.5. The cells were incubated for 20 min in a 37^oC incubator equilibrated with 95% air and 5% CO₂, and the cells were washed with HBSS (1 mL) three times.6. After trypsinization (250 µL), stop the reaction with serum medium (1 mL), 1.25 ml of the cell suspension was transferred to a microcentrifuge tube.7. The cells suspension was centrifuged at 1,500 rpm for 3 minutes.8. The supernatant was discarded and HBSS (1 mL) was added to the microcentrifuge tube and suspended by pipetting.9. The cells suspension was centrifuged at 1,500 rpm for 3 minutes and the supernatant was discarded.10. 1 μmol/L FerroOrange in MEM (serum-free medium) (300 μL) was added to the cells.11. The cells were incubated for 15 -30 min in a 37^oC incubator equilibrated with 95% air and 5% CO₂.12. The stained cells were passed through a cell strainer and analyze samples using a flow cytometer.

<Important Points>

1. FerroOrange leaks into the extracellular space due to washing cells after staining; therefore, we suggest you measure cells immediately after staining (Non wash).

2.  We recommend you add equal volumes of dye solutions, because fluorescence intensity can sometimes vary in a dye solution volume-dependent manner.

3. To verify experimental conditions for detecting Fe(II), we recommend you prepare a sample containing ammonium iron (II) sulfate and then observe changes in the fluorescence intensity of FerroOrange.