Lipid Peroxide Detection - Liperfluo

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Liperfluo

Item #	Unit Size
L248-10	1 set (50 ug x 5)

For Research Use Only Products

Application: lipid peroxide detection

Lipid peroxide selective measurement
Less photo-damage to cells
Applicable for microscopy and FCM analysis

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

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

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.

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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References

1) K. Yamanaka, Y. Saito, J. Sakiyama, Y. Ohuchi, F. Oseto and N. Noguchi, "A Novel Fluorescent Probe with High Sensitivity and Selective Detection of Lipid Hydroperoxides in Cells", RSC Adv., 2012, 2, (20), 7894.

2) V. E. Kagan, G. W. Mao, F. Qu, J. P. F. Angeli, S. Doll, C. S. Croix, H. H. Dar, B. Liu, V. A. Tyurin, V. B. Ritov, A. A. Kapralov, A. A. Amoscato, J. Jiang, T. Anthonymuthu, D. Mohammadyani, Q. Yang, B. Proneth, J. K. Seetharaman, S. Watkins, I. Bahar, J. Greenberger, R. K. Mallampalli, B. R. Stockwell, Y. Y. Tyurina, M. Conrad and H. Bayır, "Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis", Nature Chemical Biology., 2017, 13, (1), 81.

3)Y. Nakashima, S. Ohta, A. M. Wolf, "Blue light-induced oxidative stress in live skin.", Free Radical Biology and Medicine., 2017, 108, 300.

4) M. Tsugita, N. Morimoto and M. Nakayama, "SiO2 and TiO2 nanoparticles synergistically trigger macrophage inflammatory responses", Particle and Fibre Toxicology., 2017, DOI 10.1186/s12989-017-0192-6.

5) K. Iuchi, A. Imoto, N. Kamimura, K. Nishimaki, H. Ichimiya, T. Yokota and S. Ohta, "Molecular hydrogen regulates gene expression by modifying the free radical chain reactiondependent generation of oxidized phospholipid mediators", Scientific Reports., 2016, DOI: 10.1038/srep18971.

6) A. J. Clark, and H. R. Petty., "WO3/Pt nanoparticles promote light-induced lipid peroxidation and lysosomal instability within tumor cells.", Nanotechnology., 2016, 27, (7), 075103.

7) T. Otani, M. Matsuda, A. Mizokami, N. Kitagawa, H. Takeuchi, E. Jimi, T. Inai and M. Hirata, "Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300", Cell Death Dis., 2018, 9, 1194.

8) H.H. Dar, Y.Y. Tyurina, K. Mikulska-Ruminska, I. Shrivastava, H.C. Ting, V.A. Tyurin, J. Krieger, C.M. St Croix, S. Watkins, E. Bayir, G. Mao, C. Ambruster, A. Kapralov, H. Wang, M.H. Parsek, T.S. Anthonymuthu, A.F. Ogunsola, B.A. Flitter, C.J. Freedman, J.R. Gaston, T.R. Holman, J.M. Pilewski, J.S. Greenberger, R.K. Mallampalli, Y. Doi, J.S. Lee, I. Bahar, J.M. Bomberger, H. Bayır, V.E. Kagan. "Pseudomonas aeruginosa utilizes host polyunsaturated phosphatidylethanolamines to trigger theft-ferroptosis in bronchial epithelium." The journal of Clinical Investigation. 2018, DOI: 10.1172/JCI99490.

9) H. Alborzinia, T. I. Ignashkova, F. R. Dejure, M. Gendarme, J. Theobald, S. Wolfl, R. K. Lindemann and J. H. Reiling , "Golgi stress mediates redox imbalance and ferroptosis in human cells", Commun Biol.., 2018, 1, (210), DOI: 10.1038/s42003-018-0212-6.

10) W. Wang, M. Green, J. E. Choi, M. Gijon, P. D. Kennedy, J. K. Johnson, P. Liao, X. Lang, I. Kryczek, A. Sell, H. Xia, J. Zhou, G. Li, J. Li, W. Li, S. Wei, L. Vatan, H. Zhang, W. Szeliga , W. Gu, R. Liu, T. S. Lawrence, C. Lamb, Y. Tanno, M. Cieslik, E. Stone, G. Georgiou, T. A. Chan, A. Chinnaiyan, W. Zou, "CD8+ T cells regulate tumour ferroptosis during cancer immunotherapy.", Nature., 2019, 569, (7755), 270.

11) M. Gao, J. Deng, F. Liu, A. Fan, Y. Wang, H. Wu, D. Ding, D. Kong, Z. Wang, D. Peer, Y. Zhao, 'Triggered ferroptotic polymer micelles for reversing multidrug resistance to chemotherapy', Biomaterials., 2019, 233, 119486.

12) N. Wang, GZ. Zeng, JL. Yin, ZX. Bian, Artesunate activates the ATF4-CHOP-CHAC1 pathway and affects ferroptosis in Burkitt's Lymphoma, Biochem. Biophys. Res. Commun., 2019, 519, (3), 533-539.

13) T.Tsukui, Takayuki Tsukui, Z. Chen, H. Fuda, T. Furukawa, K. Oura, T. Sakurai, S. Hui, H. Chiba, 'Novel Fluorescence-Based Method To Characterize the Antioxidative Effects of Food Metabolites on Lipid Droplets in Cultured Hepatocytes', J. Agric. Food Chem., 2019, 67, (35), 9934-9941.

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

1) N. Soh, T. Ariyoshi, T. Fukaminato, H. Nakajima, K. Nakano and T. Imato, "Swallow-tailed Perylene Derivative: a new Tool for Fluorescent Imaging of Lipid Hydroperoxides", Org. Biomol. Chem., 2007, 5, 3762.
2) K. Yamanaka, Y. Saito, J. Sakiyama, Y. Ohuchi, F. Oseto and N. Noguchi, "A Novel Fluorescent Probe with High Sensitivity and Selective Detection of Lipid Hydroperoxides in Cells", RSC Adv., 2012, 2, (20), 7894.
3) V. E. Kagan, G. W. Mao, F. Qu, J. P. F. Angeli, S. Doll, C. S. Croix, H. H. Dar, B. Liu, V. A. Tyurin, V. B. Ritov, A. A. Kapralov, A. A. Amoscato, J. Jiang, T. Anthonymuthu, D. Mohammadyani, Q. Yang, B. Proneth, J. K. Seetharaman, S. Watkins, I. Bahar, J. Greenberger, R. K. Mallampalli, B. R. Stockwell, Y. Y. Tyurina, M. Conrad and H. Bayır, "Oxidized arachidonic and adrenic PEs navigate cells to ferroptosis", Nature Chemical Biology., 2017, 13, (1), 81.
4)Y. Nakashima, S. Ohta, A. M. Wolf, "Blue light-induced oxidative stress in live skin.", Free Radical Biology and Medicine., 2017, 108, 300.
5) M. Tsugita, N. Morimoto and M. Nakayama, "SiO2 and TiO2 nanoparticles synergistically trigger macrophage inflammatory responses", Particle and Fibre Toxicology., 2017, DOI 10.1186/s12989-017-0192-6.
6) K. Iuchi, A. Imoto, N. Kamimura, K. Nishimaki, H. Ichimiya, T. Yokota and S. Ohta, "Molecular hydrogen regulates gene expression by modifying the free radical chain reactiondependent generation of oxidized phospholipid mediators", Scientific Reports., 2016, DOI: 10.1038/srep18971.
7) A. J. Clark, and H. R. Petty., "WO3/Pt nanoparticles promote light-induced lipid peroxidation and lysosomal instability within tumor cells.", Nanotechnology., 2016, 27, (7), 075103.
8) T. Otani, M. Matsuda, A. Mizokami, N. Kitagawa, H. Takeuchi, E. Jimi, T. Inai and M. Hirata, "Osteocalcin triggers Fas/FasL-mediated necroptosis in adipocytes via activation of p300", Cell Death Dis., 2018, 9, 1194.
9) H.H. Dar, Y.Y. Tyurina, K. Mikulska-Ruminska, I. Shrivastava, H.C. Ting, V.A. Tyurin, J. Krieger, C.M. St Croix, S. Watkins, E. Bayir, G. Mao, C. Ambruster, A. Kapralov, H. Wang, M.H. Parsek, T.S. Anthonymuthu, A.F. Ogunsola, B.A. Flitter, C.J. Freedman, J.R. Gaston, T.R. Holman, J.M. Pilewski, J.S. Greenberger, R.K. Mallampalli, Y. Doi, J.S. Lee, I. Bahar, J.M. Bomberger, H. Bayır, V.E. Kagan. "Pseudomonas aeruginosa utilizes host polyunsaturated phosphatidylethanolamines to trigger theft-ferroptosis in bronchial epithelium." The journal of Clinical Investigation. 2018, DOI: 10.1172/JCI99490.
10) H. Alborzinia, T. I. Ignashkova, F. R. Dejure, M. Gendarme, J. Theobald, S. Wolfl, R. K. Lindemann and J. H. Reiling , "Golgi stress mediates redox imbalance and ferroptosis in human cells", Commun Biol.., 2018, 1, (210), DOI: 10.1038/s42003-018-0212-6.v
11) W. Wang, M. Green, J. E. Choi, M. Gijon, P. D. Kennedy, J. K. Johnson, P. Liao, X. Lang, I. Kryczek, A. Sell, H. Xia, J. Zhou, G. Li, J. Li, W. Li, S. Wei, L. Vatan, H. Zhang, W. Szeliga , W. Gu, R. Liu, T. S. Lawrence, C. Lamb, Y. Tanno, M. Cieslik, E. Stone, G. Georgiou, T. A. Chan, A. Chinnaiyan, W. Zou, "CD8+ T cells regulate tumour ferroptosis during cancer immunotherapy.", Nature., 2019, 569, (7755), 270.
12) M. Gao, J. Deng, F. Liu, A. Fan, Y. Wang, H. Wu, D. Ding, D. Kong, Z. Wang, D. Peer, Y. Zhao, "Triggered ferroptotic polymer micelles for reversing multidrug resistance to chemotherapy", Biomaterials., 2019, 233, 119486.
13) N. Wang, GZ. Zeng, JL. Yin, ZX. Bian, "Artesunate activates the ATF4-CHOP-CHAC1 pathway and affects ferroptosis in Burkitt's Lymphoma", Biochem. Biophys. Res. Commun., 2019, 519, (3), 533-539.
14) T.Tsukui, Takayuki Tsukui, Z. Chen, H. Fuda, T. Furukawa, K. Oura, T. Sakurai, S. Hui, H. Chiba, "Novel Fluorescence-Based Method To Characterize the Antioxidative Effects of Food Metabolites on Lipid Droplets in Cultured Hepatocytes", J. Agric. Food Chem., 2019, 67, (35), 9934-9941.
15) P. K. Mishra, A. Adameova, J. A. Hill, C. P. Baines, P. M. Kang, J. M. Downey, J. Narula, M. Takahashi, A. Abbate, H. C. Piristine, S. Kar, S. Su, J. K. Higa, N. K. Kawasaki and T. Matsui, "Guidelines for evaluating myocardial cell death", Am. J. Physiol.: Heart Circ. Physiol. ., 2019, 317, (5), H891-H922 .

Q. Which excitation filter or laser should I use for fluorescent microscope or flow cytometry?

A. Fluorescent microscope: GFP filter (ex. 450 – 490nm, em. 500 – 545nm)

FITC filter (ex. 467 – 498nm, em. 513 – 556nm)

Flow Cytometry: ex. 488nm

Q. Does phenol red or serum affect detection?

For high background or low fluorescence, is there anything I can do for improvement?

A. No, phenol red or serum will not affect detection. However, if there is high background, please use PBS instead.

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 devise setting by following: increase the excitation light strength or exposure time.

Q. Can Liperfluo be used on both suspended and adjacent cells?

Can I use it on fixed cells?

A. Yes, Liperfluo can be used on both suspended and adjacent cells.

We have data for HL-60 (suspended cells), CHO and SH-SY5Y (adjacent cells).

Liperfluo can NOT be used on fixed cells.

	Q. Can I store Liperfluo (DMSO) solution?
	A. 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.

	Q. What is recommended concentration of Liperfluo?
	A. For cell staining, we recommend concentration between 1 to 10 μM and DMSO concentration lower than 1%.