Lyso-FerroRed

Lysosomal Iron Ion Detection Reagent

Detects Fe²⁺ within the lysosomes of living cells
Enables imaging via fluorescence microscopy and quantification with plate readers and flow cytometers

Product code

L270　 Lyso-FerroRed
CAS No.

1616953-95-7
Chemical name

3'-(dimethylamino)-N,N-dimethyl-3H-spiro[isobenzofuran-1,9'-xanthen]-6'-amine oxide
MW

388.46

Unit size	Price	Item Code
35 nmol	＄380.00	L270-10

*35 nmol can be used for 17 assays at 35 mm dish (final concentration 1 µmol/l).

Order

35 nmol

Quantity may be adjusted within the cart.

C of A

SDS download

The importance of Lysosomal Iron in Ferroptosis

Ferroptosis, a type of cell death induced by iron-dependent lipid peroxidation, has been established as a factor in various diseases, including cancer, neurodegenerative diseases, and age-related disorders. While previous research primarily focused on total cellular iron content, recent studies have emphasized the importance of iron localization within organelles, particularly lysosomes. Lysosomes are known not only as sites responsible for cellular degradation and recycling, but also as storage sites for iron. Some researchers have suggested that iron dynamics within lysosomes may trigger ferroptosis*. Against this backdrop, techniques for selectively detecting and evaluating lysosomal iron have garnered significant attention.

* Y. Saimoto, et al., Nature Communications, 2025, 16, 3554.

Ferroptosis Analysis Products

Product Name	Target	Ditection Properties
FerroOrange	Intracellular Fe²⁺	Microscopy, Plate reader Ex: 543 nm / Em: 580 nm
Mito-FerroGreen	Mitochondrial Fe²⁺	Microscopy Ex: 505 nm / Em: 535 nm
Lyso-FerroRed	Lysosomal Fe²⁺	Microscopy, FCM, Plate reader Ex: 551 nm / Em: 571 nm
Iron Assay Kit -Colorimetric-	Fe²⁺ and Fe³⁺	Plate reader Colorimetric, λ: 593 nm
Liperfluo	Lipid Peroxide	Microscopy, FCM Ex: 488 nm / Em: 500-550 nm
Lipid Peroxidation Probe -BDP 581/591 C11-	Lipid Peroxidation Process	Microscopy, FCM, Plate reader Pre-reaction, Ex: 488 nm / Em: 510-550 nm Post-reaction, Ex: 561 nm / Em: 600-630 nm
MDA Assay Kit	Malondialdehyde	Plate reader Fluorescence, Ex: 540 nm / Em: 590 nm Colorimetric, λ: 532 nm
Cystine Uptake Assay Kit	Cystine uptake	Plate reader Ex: 485 nm / Em: 535 nm
GSSG/GSH Quantification Kit	GSSG and GSH	Plate reader Colorimetric, λ: 405 nm

Manual

Manual

Technical info

More than 10 years have passed since Stockwell et al. discovered ferroptosis, which is caused by an iron ion-dependent accumulation of lipid peroxides. It has become clear that ferroptosis is a form of cell death, in which free iron (Fe²⁺) generates reactive oxygen species (ROS) through the Fenton reaction¹⁾, which in turn oxidize lipids. More recently, it has been reported that lipid oxidation damages lysosomal membranes and causes iron to leak from lysosomes, thereby spreading it to other cellular organelles²⁾. Lyso-FerroRed can detect intracellular lysosomal free iron (Fe²⁺). Lyso-FerroRed penetrates the cell membrane and selectively reacts with Fe²⁺ in lysosomes, emitting strong fluorescence. Reaction between Lyso-FerroRed and Fe²⁺ is irreversible and differs from those based on the detection principle of calcium iron probes such as Fluo 3. Lyso-FerroRed has no chelating ability.

1) S. J. Dixon, et al., Cell, 2012, 149, 1060–1072
2) Y. Saimoto, et al., Nature Communications, 2025, 16, 3554

[Iron Detection Principle of Lyso-FerroRed]

This product was developed under the guidance of Professor Tasuku Hirayama of Gifu Pharmaceutical University.

Selection of Iron Detection Reagents

Select the appropriate reagent based on your experimental method and detecting equipment.

	Lyso-FerroRed	FerroOrange	Mito-FerroGreen	Iron Assay Kit -Colorimetric-
Intracellular localization	Lysosome	Intracellular	Mitochondria	ー (Tissue)
Detection properties	Fluorometric λex : 551 nm, λem : 571 nm	Fluorometric λex : 543 nm, λem : 580 nm	Fluorometric λex : 505 nm, λem : 535 nm	Colorimetric 593 nm
Supported devices （filter）	Microscope, Plate reader, Flow cytometry(TRITC)	Microscope, Plate reader(Cy3)	Microscope(FITC, GFP)	Plate reader
Target	Lysosomal Fe²⁺ in live cells	Intracellular Fe²⁺ in live cells	Mitochondrial Fe²⁺ in live cells	Fe²⁺ and Fe³⁺ in tissue
Usage count	35 nmol can be used for 17 assays at 35 mm dish (final concentration 1 µmol/l).	1 tube (24 µg) can be used for 17 assays at 35 mm dish (final concentration 1 µmol/L)	1 set (50 µg x 2) can be used for 10 assays at 35 mm dish (final concentration 5 µmol/L)	50 tests (Make standard curve and detect Fe²⁺ only: 4 samples, detect Fe²⁺ and Fe³⁺: 2 samples)

Detection and Quantification of Lysosomal Fe²⁺

We confirmed changes in intracellular Fe²⁺ within lysosomes of LPS-treated HT-1080 cells using Lyso-FerroRed. We achieved this by varying the presence or absence of the iron-chelating reagent Bpy (final concentration: 1 mmol/l) and by adding iron (ferrous ammonium sulfate, final concentration: 100 μmol/l).

Fluorescence microscopy imaging

[Detection condition] Ex/Em = 564 nm / 565-620 nm

Quantification by plate assay

[Detection condition] Ex/Em = 550 nm / 580 nm

Detection by flow cytometry

[Detection condition] Ex/Em = 565 nm / 565-617 nm

High Selectivity for Fe²⁺

One milliliter of 50 mmol/l HEPES buffer (pH 7.4) was mixed with 2 µl of 1 mmol/l Lyso-FerroRed and various metals. After reacting at room temperature for one hour, the fluorescence intensity was measured.

[Final concentration]
Lyso-FerroRed：2 μmol/l
Mn, Co, Ni, Fe²⁺, Fe³⁺, Zn, Cu : 20 nmol/l
Na, K, Mg, Ca : 1 mmol/l
Bpy : 100 µmol/l

[Detection conditions]
Ex: 550 nm, Em: 580 nm

Definite Lysosomal Localization

To confirm the lysosomal localization of Lyso-FerroRed, we performed co-staining with various organelle-staining reagents and confirmed that Lyso-FerroRed selectively stains lysosomes.

Lysosome staining: LysoPrime Deep Red (Product Code: L264)
Lipid droplet staining: Lipi-Deep Red (product code LD04)
Mitochondria staining: MitoBright LT Deep Red (product code MT12)

*Lyso-FerroRed is displayed in pseudo-color to make it easier to visualize the merged images.

Fluorescence Property

Experimental example: Changes in Each Indicator Following the Co-treatment of Ferroptosis Inducers and Lysosomal Inhibitors

Previous studies have suggested that ferrotosis susceptibility varies among cancer cell lines. It has also been reported that increasing lysosomal stress in ferrotosis-resistant cancer cells can promote ferrotosis*. We treated A549 cells, which exhibit ferroptosis resistance, with the ferroptosis inducer RSL3 or RSL3 combined with the lysosomal inhibitor chloroquine (CQ) for 24 hours. Then, we analyzed changes in cell viability, lysosomal Fe²⁺, and lysosomal content. Treatment with RSL3 alone did not significantly alter cell viability or intracellular Fe²⁺ levels; however, some lysosomal aggregation (strong LysoPrime Deep Red signal) was observed. In contrast, cells treated with both RSL3 and CQ simultaneously exhibited increased intracellular Fe²⁺ levels, lysosomal enlargement, and decreased cell viability, which is consistent with previous reports. These results suggest that increased intracellular Fe²⁺ may promote ferroptosis.

* Saimoto. Y, et al., Nature Communications, 2025, 16, 3554.

[Products in use]
Cell viability: Cell Counting Kit-8 (Product code: CK04)
Lysosomal Fe²⁺: Lyso-FerroRed (Product code: L270)
Lysosomal mass: LysoPrime Deep Red - High Specificity and pH Resistance (Product code: L264)

References

Open References

No.	Sample	Instruments	Publications
1)	Cell （HepG2）	Fluorescence microscope	Niwa, M., Hirayama, T., Okuda, K., Nagasawa, H., "A New Class of High-Contrast Fe(II) Selective Fluorescent Probes Based on Spirocyclized Scaffolds for Visualization of Intracellular Labile Iron Delivered by Transferrin", Org. Biomol. Chem. 2014, 12 (34), 6590–6597. DOI: 10.1039/C4OB00935E *The compound referred to as “HMRhoNox-M” in the paper is “Lyso-FerroRed”.
2)	Cell （Calu-1）	Fluorescence microscope	Saimoto, Y., Kusakabe, D., Morimoto, K., Matsuoka, Y., Kozakura, E., Kato, N., Tsunematsu, K., Umeno. T., Kiyotani, T., Matsumoto, S., Tsuji, M., Hirayama, T., Nagasawa. H., Uchida, K., Karasawa, S., Jutanom, M., Yamada, K., "Lysosomal lipid peroxidation contributes to ferroptosis induction via lysosomal membrane permeabilization", Nat Commun, 2025, 16, 3554. DOI: 10.1038/s41467-025-58909-w *The compound referred to as “Lyso-RhoNox” in the paper is “Lyso-FerroRed.”

Q & A

Q How many samples can be measured by L270?: A

The number of measurable samples is as follows:
・ 96-well plate: 3 plates
・ ibidi 8-well plate: 21 plates
・ 35 mm dish: 17 dishes
・ 6-well plate: 17 wells

Q Are there any experimental examples that serve as positive controls?: A

The manual includes examples of detection using HT-1080 cells incorporating ammonium iron sulfate. Please refer to steps 1-6 in the experimental examples section.

Q Are there any precautions to take when preparing the working solution?: A

Prepare using HBSS or serum-free medium. Preparing in serum-containing medium causes the iron in the serum to react with the dye, resulting in increased background fluorescence.

Q Are there any precautions to take when detecting with a flow cytometer?: A

Typically, trypsin is used to detach cells from dishes after staining with low-molecular-weight fluorescent probes. After detaching cells with trypsin, cells are recovered using serum-containing medium to quench residual trypsin.
Lyso-FerroRed reacts with iron in serum when it comes into contact with serum-containing medium. Therefore, when detaching cells, scrape them off using a scraper and recover them in PBS or HBSS.

Q Is this dye compatible with fixed cells?: A

No.
Lyso-FerroRed signal is undetected and the changes in intracellular iron(II) ions cannot be confirmed in fixed cells.

HT-1080 cells treated with ammonium iron(II) sulfate (final concentration: 100 μmol/L) were stained with Lyso-FerroRed, then observed under a fluorescence microscope after either no fixation or 30 minutes of fixation (4% PFA/PBS).

[Dtection condition]
Ex : 564 nm(0.8 %), Em : 570-620 nm
Gain : 480 V

Handling and storage condition

Specification
Fluorescence spectrum：	To pass test
Confirmation test：	To pass test

Handling and storage condition
Ambient temperature, Protect from light

SDS download

Lyso-FerroRed

Lyso-FerroRed

Lysosomal Iron Ion Detection Reagent

The importance of Lysosomal Iron in Ferroptosis

Manual