Lysosomal Lipid Radical Probe -Lyso-NBD-Pen-

Lysosomal Lipid Radical Probe -Lyso-NBD-Pen-

Lysosomal Lipid Radical Detection

  • Enables detection of lipid radicals in lysosomes of live cells
  • Compatible with fluorescence microscopy and flow cytometry for detection and quantification
  • Detects changes in lipid localization after reaction through binding to lipid radicals
  • Product code
    L271　 Lysosomal Lipid Radical Probe -Lyso-NBD-Pen-
Unit size Price Item Code
2 nmol ＄380 L271-10

*2 nmol can be used for 10 assays in 35 mm dishes (final concentration: 0.1 µmol/l).

Order
2 nmol
Quantity may be adjusted within the cart.

The Importance of Lipid Radicals in Ferroptosis

Ferroptosis is a form of cell death triggered by iron-dependent lipid peroxidation and has been increasingly implicated in various diseases, including cancer, neurodegenerative diseases, and age-related disorders. Lipid radicals are key intermediates involved in the propagation of lipid peroxidation chain reactions and are attracting attention as indicators of early changes and progression of lipid oxidation during ferroptosis. In recent years, ferroptosis has also been reported to be initiated by lysosomal lipid peroxidation, highlighting the importance of detecting lipid radicals in lysosomes.¹⁾

Lyso-NBD-Pen localizes to lysosomes and enables specific detection of lipid radicals generated within lysosomes. Simply add the reagent to cultured cells to detect lysosomal lipid radicals using fluorescence microscopy or flow cytometry.

1) S.Saimoto, et al., Nat. Commun., 2025, 16, 3554. 

 

This product was developed under the guidance of Professor Kenichi Yamada of Kyushu University.

Product Name Target Detection 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 II GSSG and GSH Plate reader
Colorimetric, λ: 405 nm

Manual

Technical info

Please select the appropriate reagent based on your experimental method and detection instrument used for lipid peroxidation analysis.

  Liperfluo [This Product] Lysosomal Lipid Radical Probe -Lyso-NBD-Pen- Lipid Radical Probe -NBD-Pen- Lipid Peroxidation Probe -BDP 581/591 C11-
Target Lipid peroxides Lipid radicals Lipid radicals Lipid radicals + hydroxyl radicals

Localization

 Intracellular

Lysosomal

Intracellular

 Intracellular

Compatible Instruments

Fluorescence microscopy,
​Flow cytometry

Fluorescence microscopy,
​Flow cytometry

 Fluorescence microscopy,
Flow cytometry

Fluorescence microscopy,
Flow cytometry,
Plate reader

Detection Conditions
(Fluorescence Microscopy)

Fluorescence
Ex: 488 nm, Em: 500–550 nm

 Fluorescence
Ex: 488 nm, Em: 490–600 nm		 Fluorescence
Ex: 488 nm, Em: 490–600 nm

Fluorescence
Green: Ex: 488 nm, Em: 510–550 nm
Red: Ex: 561 nm, Em: 600–630 nm
Sensitivity ☆ ☆☆ ☆☆☆

Interaction with Target

Reacts only
(does not bind)

 

Reacts and binds

 Reacts only
(does not bind)
Features Specifically detects lipid peroxides.

Binds to lipid radicals, enabling detection of changes in lipid localization after the reaction.

 Ratiometric detection enables quantification using a plate reader.

High Selectivity for Lipid Radicals

The reactive oxygen species listed below were mixed with this reagent in 100 mmol/l phosphate buffer (pH 7.4), and the fluorescence intensity was measured after a 30-minute reaction.

Reliable Lysosomal Localization

To confirm the lysosomal localization of this probe reagent, co-staining was performed with the lysosome staining reagent LysoPrime Deep Red (Product Code: L264). The fluorescence signals of both reagents were colocalized, confirming that Lyso-NBD-Pen selectively stains lysosomes.

Detection of Lysosomal Lipid Radicals

Treatment with the ferroptosis inducer RSL3 resulted in an increase in lipid radicals within lysosomes. Furthermore, co-treatment with the lipid radical scavenger Liproxstatin-1 (Lip-1) resulted in a decrease in the signal, indicating that this reagent selectively detects lipid radicals.

Quantification of Lysosomal Lipid Radicals

HT1080 cells were treated with the ferroptosis inducer RSL3, and the increase in lysosomal lipid radicals was quantified by flow cytometry.

Fluorescence Properties

Experimental Example: Comparison of Lysosomal Lipid Radical Detection in Ferroptosis-Sensitive and -Resistant Cells

Changes in lysosomal lipid radicals were detected in HT-1080 cells, which are highly sensitive to ferroptosis, and A549 cells, which are resistant to ferroptosis, following treatment with RSL3, a ferroptosis inducer.

In A549 cells, a ferroptosis-resistant cell line, no significant difference in fluorescence intensity was observed compared with the control, even when the RSL3 treatment concentration and duration of RSL3 exposure were increased (concentration: up to 2.5 μmol/l; duration: up to 3 hours). In contrast, in HT-1080 cells, a ferroptosis-sensitive cell line, treatment with 1 μmol/L RSL3 for 2 hours increased fluorescence derived from lysosomal lipid radicals and caused changes in their localization.

These results demonstrate that this product can detect differences in lysosomal lipid radical generation and localization changes depending on the cell type.

 

Q & A

Q

How many samples can be measured with this product?

A

The number of samples that can be measured with 2 nmol of this product is as follows:

  • 96-well plate: 2 plates
  • ibidi 8-well plate: 100 wells
  • 35 mm dish: 10 dishes
  • 6-well plate: 10 wells

Q

Is there an experimental example that can be used as a positive control?

A

Please refer to the instruction manual for the lysosomal lipid radical detection experiment using HT-1080 cells treated with RSL3 to induce ferroptosis.
If endogenous lipid radicals are detected, they can be scavenged by co-treatment with Liproxstatin-1.

Q

Can Lyso-NBD-Pen be used for observation after cell fixation following staining?

A

No. This product is suitable only for live-cell imaging, as the fluorescence signal decreases after fixation.

Q

What precautions should be taken when observing samples with a fluorescence microscope?

A

NBD, the fluorescent scaffold of this probe, undergoes photobleaching when continuously exposed to excitation light.
When acquiring imaging data, adjust the focus under bright-field observation before acquiring fluorescence images.

Q

How long can samples be observed after staining?

A

We have confirmed that samples can be observed for up to 24 hours after staining.

Handling and storage condition

Specification
Appearance： Orange to red solid
Solubility in Dimethyl sulfoxide： To pass test
Dye content： To pass test
Handling and storage condition
Store at room temperature.
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