Liperfluo, a perylene derivative containing oligooxyethylene, is designed and exclusively developed by Dojindo for the detection of lipid peroxides. Liperfluo emits intense fluorescence by lipid peroxide specific oxidation in organic solvents such as ethanol. Among fluorescent probes that detect Reactive Oxygen Species(ROS), Liperfluo is the only compound that can specifically detect lipid peroxides. Since the excitation and emission wavelengths of the oxidized Liperfluo are 524 nm and 535 nm, respectively, photo-damage and auto-fluorescence from the sample can be minimized. The tetraethyleneglycol group linked to one end of diisoquinoline ring helps its solubility and dispersibility to aqueous buffer. Liperfluo’s oxidized form is nearly nonfluorescent in an aqueous media and emits a strong fluorescence in lipophilic sites such as in cell membranes. Therefore it can easily be applied to lipid peroxide imaging by a fluorescence microscopy and a flow cytometric analysis for living cells. Liperfluo is used to monitor lipid peroxidation in ferroptosis research.

Reaction of Liperfluo with lipid peroxide

Properties of Liperfluo

Live cell imaging of lipid peroxide

Procedure1. Innoculate SH-SY5Y cells(6.0 x 105 cells/well) to a 6-well plate.2. Incubate the plate at 37 ºC for overnight.3. Add Liperfluo, DMSO solution (final conc. 20 μM) and incubate at37 ºC for 15 min.4. Add either Cumene Hydroperoxide (final conc. 100 μM) or AIPH*(final conc. 6 mM).5. Incubate at 37 ºC for 2 hours.6. Observe fluorescent by microscope**.* AIPH: 2,2 Eazobis-[2-(2-imidazolin-2-yl)propane]dihydrochloride** Olympus IX-71 epifluorescent microscope, mirror unit: U-MNIBA3, exposure time: 10 sec, ISO: 800

Data was kindly provided from Dr. N. Noguchi, Doshisha University, System Life Science Laboratory.

Flow cytometric analysis of lipid hydroperoxides in live cell

Procedure1. Innoculate SH-SY5Y cells (6.0 x 105 cells/well) to a 6-well plate.2. Incubate the plate at 37 ºC for overnight.3. Add Liperfluo, DMSO solution (final conc. 20 μM) and incubate at 37 ºC for 15 min.4. Add either Cumene Hydroperoxide (final conc. 100 μM) or AIPH*(final conc. 6 mM).5. Incubate at 37 ºC for 2 hours.6. Wash cells with PBS.7. Collect cells with PBA and analyse by flow cytometer**.* AIPH: 2,2 Eazobis-[2-(2-imidazolin-2-yl)propane]dihydrochloride** BD FACSAriaTM I

Data was kindly provided from Dr. N. Noguchi, Doshisha University, System Life Science Laboratory.

Lipid peroxide of living cells Cell line: L929Microscope: Zeiss LSM510METAFilter type: FITC（GFP, Alexa488）wide filterHFT UV/488NFT490BP505-550

Procedure:1. Prepare cell suspension (2.5 x 10⁵ cell/well) in 35mm Glass bottom dish and incubate at 37^oC overnight in CO₂.2. Discard the media and add new media containing Liperfluo (final conc. 1μM) .3. Incubate at 37^oC for 30 min in CO₂.4. Discard the media add new media containing t-BHP (final conc. 250μM ).5. Incubate at 37^oC for 2 hours in CO₂.6. Observe using confocal microscope.Data was kindly provided from Dr. T. Kumagai and Dr. H. Imai, Kitasato University, School of Pharmacy.

Necrosis, apoptosis and autophagy is known as cell death-related processes. 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. Liperfluo is used as a fluorescent prove which can detect intracellular lipid peroxide directly.

Ferroptosis: A Regulated Cell Death Nexus Linking Metabolism, Redox Biology, and Disease.B. R. Stockwell et al., Cell, 2017, 171(2), 273.

Oxidized Arachidonic/Adrenic Phosphatidylethanolamines Navigate Cells to FerroptosisV. E. Kagan et al., Nat. Chem. Biol., 2017, 13, (1), 81.

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