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
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 addition |
Stimulation |
Tips |
| After stimulation |
Erastin
(overnight) |
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 stimulation |
t-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
|
Procedure
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
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Data was kindly provided from Dr. N. Noguchi, Doshisha University, System Life Science Laboratory.
Flow cytometric analysis of lipid hydroperoxides in live cell
|
Procedure
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 cells
Cell line: L929
Microscope: Zeiss LSM510META
Filter type: FITC（GFP, Alexa488）wide filter
HFT UV/488
NFT490
BP505-550
|
Procedure:
- Prepare cell suspension (2.5 x 105 cell/well) in 35mm Glass bottom dish and incubate at 37 oC overnight in CO2.
- Discard the media and add new media containing Liperfluo (final conc. 1μM) .
- Incubate at 37 oC for 30 min in CO2.
- Discard the media add new media containing t-BHP (final conc. 250μM ).
- Incubate at 37 oC for 2 hours in CO2.
- 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.
B. R. Stockwell et al., Cell, 2017, 171(2), 273.
V. E. Kagan et al., Nat. Chem. Biol., 2017, 13, (1), 81.
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