Updated: February 15, 2019
For research use only

FerroOrange
Table 1 Product information
CAT. No.

Product namename

GC904-01Size
35 nmol × 5

FerroOrange
GC904-02

1.

35 nmol × 1

Storage upon receipt

Stability≤−20°C, keep desiccatedand protected from light.

1 year (when unopenedand stored as described.)

About FerroOrange

FerroOrange is an orange fluorescent probe for cell imaging designed to detect specifically labile iron (II)ions (Fe2+) localized in endoplasmic reticulum (ER). This reagent reacts specifically to Fe 2+ by irreversibly
changing to an orange fluorescent substance. Therefore, this probe can be used to selectively distinguishFe2+ ions from other metal ions.

Fig.1 Absorption spectrum and fluorescence spectrum of FerroOrange in 0.05 M HEPES buffer (pH7.4). The absorption maximum is observed at 542 nm, and the fluorescence maximum isobserved at 572 nm.

■ Storage
This product is shipped at room temperature. Upon receipt, store the product desiccated and protected fromlight at ≤ −20°C. Storing as a solution is not recommended.

Spin beforeopeningOpen at roomtemperature

Store in a freezerKeep dry

Store in a darkplace12.

Preparation of reagent■ Materials required but not provided
・Dimethyl Sulfoxide (DMSO)
・Appropriate washing and observation buffer (PBS pH 7.4, HBSS, etc.). It should be a solution without
phenol red.
■ Preparation of reagent
FerroOrange is a colorless powder. Return the vial to room temperature taking care not to absorb moisture,
place it into a microcentrifuge, collect the powder adhering to the lid and walls on the bottom and open thevial.
Add 35 µL of DMSO to 1 vial (35 nmol) and repeat pipetting 5 or more times to completely dissolve thereagent. The resulting solution is 1mM. The FerroOrange solution is almost colorless. Use neutral buffer
or culture medium for dilution of the DMSO solution and use immediately upon dilution. FerroOrange could
be oxidized in acidic solutions.

3.

Example of Cell Staining

Observation of labile iron (II) ions (Fe2+) in HepG2 cells
1. Seed HepG2 cells in a glass bottom dish and culture overnight.
2. Remove the culture medium from the dish and rinse cells gently twice with washing buffer to removeextracellular Fe2+.
3. Dilute 1mM stock solution of FerroOrange in HBSS to prepare a staining solution with a final
concentration of 1μM.
4. Add the staining solution to the culture vessel and incubate at 37°C for 30 minutes.
5. After the staining, rinse the stained cells twice with washing buffer, replace it with observation buffer.
6. Observe the cells with a fluorescent microscope.
※ You can detect the increase of labile Fe2+ ions as a positive control, if you added Fe2+ in HBSS orserum-free medium. For this purpose, dissolve Fe(NH4)2(SO4)2 (FAS) in pure water to prepare a
100 mM solution just before use and dilute it with serum-free cell culture medium to a final
concentration of 100 µM with HBSS. After cells were cultured in the FAS solution for 30 minutes,
wash the cells to remove extracellular FAS and add FerroOrange solution to detect intracellularFe2+.
※ After incubating the cells in this solution for 30 minutes, intracellular Fe2+ concentration rises. Wash
extracellular Fe2+ with washing buffer and add FerroOrange.
※ Do not use the solutions with serum. Because FerroOrange reacts with Fe 2+ in serum before it
reacts with Fe2+ in the cells, the intracellular Fe2+ cannot be detected correctly.
※ We recommend optimizing dye concentrations and incubation time in your conditions. In GORYO
Chemical, incubation in 1 µM dye at 37°C for 30 minutes gave good results for HeLa cells (humancervical cancer cell line). If cells tend to come off from the dish easily, usage of poly-L-lysine orother coating materials before seeding the cells is recommended.

■ Fluorescence observation
The fluorescence can be observed in a fluorescence microscope with a general G excitation filter such asfor Cy3. 532 nm, 514 nm or 561 nm lasers are often used for excitation in laser microscopes and flowcytometers. Excitation with 488 nm laser is also capable. The fluorescence emission is 572 nm. For
analysis by flow cytometer, a filter used for phycoerythrin (PE) is appropriate.
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* We are grateful to Dr. Hideko Nagasawa and Dr. Tasuku Hirayama (Gifu Pharmaceutical University) with
their support and advices for the commercialization of FerroOrange.
Table 2. Related products
Catalog noMaterialUsageGC901

FeRhoNox™-1

To detect Fe2+ localized in Golgi.

GC902

CopperGREEN™

To detect Cu+

GC903-01

FerroFarRed™

To detect Fe2+ localized in ER. Deep red fluorescent reagent.

SK2001-01

ZnAF-2

SK2002-01

ZnAF-2 DA

SK3001-01

HPF

To detect hydroxyl radicals (・OH) and peroxynitrite (ONOO-).

SK3002-01

APF

To detect hydroxyl radical (・OH), peroxynitrite (ONOO-), andhypochlorous acid (HClO).

SK3003-01

NiSPY-3

GC3004-01

OxiORANGE™

GC3006-01

HySOx

GC3007-01

HYDROP™

A101-01MAR

To detect Zn2+
To detect intracellular Zn2+

To detect peroxynitrite (ONOO-).
To detect hydroxyl radicals (・OH) and hypochlorous acid (HClO).
Orange fluorescent reagent.
To detect intracellular hypochlorous acid (HClO).
Fluorescence probe to detect only intracellular hydrogen peroxide(H2O2).
To detect hypoxic response of cells.

GORYO Chemical, Inc.
EAREE building 5F, Kita 8 Nishi 18-35-100 Chuo-ku, Sapporo 060-0008,
Japan
TEL: +81-11-624-5860
URL: https://www.goryochemical.com/en/

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