– Selective measurement of Lipid Peroxide
– Less cellular photo-damage
– Applicable for microscopy and FCM analysis

Application: lipid peroxide detection

Chemical Name: N-(4-Diphenylphosphinophenyl)-N‘-(3,6,9,12-tetraoxatridecyl)perylene-3,4,9,10-tetracarboxydiimide

Appearance: Dark red crystalline powder or solid
Purity: ≥90.0% (HPLC)
MW: 840.85, C51H41N2O8P

Storage Condition: 0-5ºC, protect from light
Shipping Condition: ambient temperature

Beginner’s Guide to Cellular Metabolism Ferroptosis and Reagent Selection Guide LDs/Mitochondria/Senescence Map Selection Guide
DescriptionReferencesQ & AManualS.D.S
Product Description
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

Reaction of Liperfluo with lipid peroxide

Properties of Liperfluo
Properties of Liperfluo

The timing for Liperfluo addition depends on the type of drug treatment.
The table below summarizes the timing for typical positive controls (Erastin/t-BHP).
Please read the manual carefully for detailed procedure.

Dye additionStimulationTips
After stimulationErastin
The fluorescence signal of Liperfluo gradually increases by autooxidation during the dye incubation. Thus, Liperfluo should be added after stimulation to reduce autooxidation of Liperfluo.
Before stimulationt-BHP
(60 min)
In the case of experimentation with t-BHP, we recommended adding Liperfluo before stimulation, because longer incubation with t-BHP results in cell damage. When Liperfluo is added before stimulation, control cells without t-BHP stimulation are required to subtract background signal.

Live cell imaging of lipid peroxide

Live cell imaging of lipid peroxideProcedure
1. 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. 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

Flow cytometric analysis of lipid hydroperoxides in live cellProcedure
1. 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 cellsLipid peroxide of living cells

Cell line: L929
Microscope: Zeiss LSM510META
Filter type: FITC(GFP, Alexa488)wide filter
HFT UV/488

1. Prepare cell suspension (2.5 x 105 cell/well) in 35mm Glass bottom dish and incubate at 37oC overnight in CO2.
2. Discard the media and add new media containing Liperfluo (final conc. 1μM) .
3. Incubate at 37oC for 30 min in CO2.
4. Discard the media add new media containing t-BHP (final conc. 250μM ).
5. Incubate at 37oC 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.

Lipid Peroxides in the process of Ferroptosis
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. C11-BOPIDY (ex. 581 nm, em. 591 nm) detects activation of ferroptosis, but it does not directly reveal the production of lipid hydroperoxides. Dojindo’s Liperfluo (ex. 524 nm, em. 535 nm), however, interacts directly with (phosphor)lipid hydroperoxides. Due to this difference, Liperfluo fluorescence more reliably indicates lipid hydroperoxide accumulation.

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 Ferroptosis
V. E. Kagan et al., Nat. Chem. Biol., 2017, 13, (1), 81.

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Which excitation filter or laser should I use for fluorescent microscope or flow cytometry?
Fluorescent microscope: GFP filter (ex. 450 – 490nm, em. 500 – 545nm)
FITC filter (ex. 467 – 498nm, em. 513 – 556nm)
Flow Cytometry: ex. 488nm
Does phenol red or serum affect detection?
No, phenol red or serum will not affect detection. However, if there is high background, please use PBS instead.
For high background or low fluorescence, is there anything I can do for improvement?
If the background is high, Liperfluo may be oxidized by light. Please avoid light during incubation by covering the solution with aluminum foil.

Increasing reaction time because of weak fluorescence will NOT improve the result due to increasing the background. Therefore, please adjust the device setting by following: increase the excitation light strength or exposure time.

Can Liperfluo be used on both suspended and adherent cells? Fixed Cells?
Yes, Liperfluo can be used on both suspended and adherent cells.
We have data for HL-60 (suspended cells), CHO and SH-SY5Y (adherent cells).
Liperfluo can NOT be used on fixed cells.
Can I store Liperfluo (DMSO) solution?
No, Liperfluo in DMSO solution can NOT be stored due to instability of Liperfluo in light. After preparing the solution, please avoid light by using aluminum foil and use it within that day.
What is recommended concentration of Liperfluo?
For cell staining, we recommend concentration between 1 to 10 μM and DMSO concentration lower than 1%.

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