Updated: November 22, 2018
For research use only

FerroFarRed™
Table 1 Product Information
Catalog no.

Product nameAmount

Storage upon receipt

Stability

GC903-01

FerroFarRed

50 nmol x 5≤−20°C, keep desiccatedand protected from light.

1 year (when unopenedand stored as described.)

1. About FerroFarRed
FerroFarRed (also known as SiRhoNox-1 or ER-SiRhoNox) is a fluorescent probe that specifically detects
labile iron (II) ions (Fe2+) via far-red fluorescence. This probe could react with only Fe2+ separately from othermetal ions and irreversibly turns into a far-red fluorescent substance. It mainly localizes inside endoplasmic
reticulum (ER)Fig. 1 Absorption spectra (left) and fluorescent spectra (right) of FerroFarRed under
0.05 M HEPES buffer (pH 7.4).
Spectrum of FerroFarRed with 100 µM Fe2+ (solid lines) and without Fe2+ (dottedlines). The absorption maximum is 646 nm, whereas the maximum fluorescencewavelength is 662 nm.
■ Storage
Upon receipt, store the product desiccated and protected from light at ≤ −20°C. Storing as a solution is notrecommended.

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Preparation of reagent■ Materials required but not provided· Dimethyl sulfoxide (DMSO)· Appropriate observation buffer (PBS pH 7.4, HBSS, etc.). It should be a solution without phenol red.
· Serum-free cell culture medium (D-MEM, etc.)■ Preparation of reagent
FerroFarRed is a blue solid. Before opening the cap, warm the vial to the room temperature. Then, use amicrocentrifuge to spin down the solid that might adhere on the cap.
Add 50 μL of DMSO to 1 vial (50 nmol) to prepare 1 mM concentration. Finally, dissolve the solid entirely
by pipetting for more than 5 times. The FerroFarRed solution is almost colorless (faint blue).

3.

Example of Cell Staining

Observation of labile Iron (lI) ions (Fe2+) in HeLa Cells
1. Seed the cells in a glass bottom dish and culture overnight.
2. Remove the culture medium from the dish and rinse twice gently with the observation buffer.
3. Dilute 1 mM stock solution of FerroFarRed in a serum-free cell culture medium to prepare a staining
solution with a final concentration of 5 μM.
4. Add the staining solution to the dish and incubate for 1 hour at 37°C.
5. After the staining, wash once with the observation buffer and replace with the observation buffer.
6. Observe the cells with a fluorescence microscope.
※ Optimal concentration of FerroFarRed and reaction time may vary on cell type and condition. InGORYO Chemical, Inc., incubating HepG2 cells with the dye under the same condition, gave goodresults. If cells are easily detached from the dish, usage of poly-L-lysine or other coating materials
is recommended.
※ You can detect the increase of labile Fe2+ if you added Fe2+ in the medium. For this purpose, dissolve
Fe(NH4)2(SO4)2(FAS) to prepare a 100 mM solution just before use, then dilute the FAS solution withserum-free cell culture medium to prepare 100 µM FAS solution. After cells were cultured in the FAS
solution for 30 minutes, wash the cells to remove extracellular FAS and add FerroFarRed solution todetect intracellular Fe2+.
※ In GORYO Chemical, Inc., dilution Fe(NH4)2(SO4)2 and FerroFarRed with HBSS, instead of serumfree medium, gave good results.
※ Do not use the solutions with serum. Under the condition, intracellular Fe2+ cannot be detected
correctly because FerroFarRed reacts with Fe2+ in the serum before reacting with intracellular Fe2+.
Measurement of Fe2+ in HepG2 Cells Using a Flow Cytometer
1. Seed the cells in a multi-well plate and culture overnight.
2. Remove the culture medium from the plate and rinse twice gently with the observation buffer.
3. Dilute 1 mM stock solution of FerroFarRed in a serum-free cell culture medium to prepare a staining
solution with a final concentration of 5 μM.
4. Add the staining solution to the plate and incubate for 1 hour at 37°C.
5. After the staining, wash once with PBS and then add 0.25% trypsin-EDTA solution to detach the cells
from the multi-well plate.
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6. Dilute 0.25% trypsin-EDTA solution with PBS on ice and centrifuge the cell suspension at 500 x g for 5
minutes to precipitate the cells.
※ Do not neutralize trypsin with serum. In the case of neutralizing with serum, intracellular Fe2+ cannot
be detected correctly because FerroFarRed reacts with Fe2+ in the serum before reacting withintracellular Fe2+.
7. Discard the supernatant and resuspend the cells in PBS.
8. Filter the cell suspension through a cell strainer (40 µm nylon mesh) to remove debris.
9. Analyze the sample using a flow cytometer.
※ Optimal concentration of FerroFarRed and reaction time may vary on cell type and condition.
※ You can detect the increase of labile Fe2+ if you added Fe2+ in the medium. For this purpose, dissolve
Fe(NH4)2(SO4)2(FAS) to prepare a 100 mM solution just before use, then dilute the FAS solution withserum-free cell culture medium to prepare 100 µM FAS solution. After cells were cultured in the FAS
solution for 30 minutes, wash the cells to remove extracellular FAS and add FerroFarRed solution todetect intracellular Fe2+.
■ Fluorescence observation
For laser excitation, wavelength around 635 nm is appropriate. The fluorescence could be detected at around
660 nm. For observation by fluorescent microscopes, use red excitation filter set for Cy5. For analysis by flow
cytometer, filter used for Allophycocyanin (APC) is appropriate.
■ References
See also the publications below, for other usages and protocols.
T. Hirayama, A. Miki, H. Nagasawa (2018) Metallomics. in press

DOI: 10.1039/c8mt00212f

K. Sakamoto, T. Suzuki, K. Takahashi, T. Koguchi, T. Hirayama, A. Mori, T. Nakahara, H. Nagasawa,
K. Ishii (2018) Exp. Eye Res. 171: 30-36 DOI: 10.1016/j.exer.2018.03.008
T. Hirayama, H. Tsuboi, M. Niwa, A. Miki, S. Kadota, Y. Ikeshita, K. Okuda, H. Nagasawa (2017) Chem.
Sci. 8: 4858-4866 DOI: 10.1039/c6sc05457a

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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://goryochemical.com/4