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Acrolein Detection Reagent in Live Cells AcroleinRED | Funakoshi

Date:October 01 2018Web Page No:81137

Funakoshi Co.,Ltd.


AcroleinRED logo

AcroleinRED reacts specifically with acrolein, an unsaturated aldehyde that exhibits strong toxicity, and visualizes it with red fluorescence. This reagent does not react with other unsaturated aldehydes or lipid metabolites etc., and enables easy detection and relative quantification of endogenous acrolein or acrolein produced by external stimuli in living cells.
This product has been commercialized with the support of Biofunctional Synthetic Chemistry Laboratory, Cluster for Pioneering Research, RIKEN.

What is Acrolein

Acrolein (2-propenal), the simplest α,β-unsaturated aldehyde, which is found in a common dietary and environmental pollutant such as tobacco smoke, is known as a highly toxic metabolite for cells. Not only in outer environment, but also acrolein is endogenously gererated in cells, especially under the oxidative stress condition. Enzymatic oxidation of threonine and polyamine species, reactive oxygen species (ROS)-mediated oxidation of highly unsaturated lipids are major source of acrolein in cells.

acrolein structure

Structural formula of Acrolein

Recent evidences indicate acrolein is more toxic to cells than ROS such as hydrogen peroxide (H2O2) or hydroxyl radicals. Because of its highly reactivity, acrolein in the cell immediately reacts with various biomolecules such as DNA/RNA and proteins, and subsequently impairs their functions. Acrolein has being researched for more than half-century and many detection methods were developed, such as fluorometric method, mass spectromy or antibody based detection method, etc. However, conventional methods are not appropriate for the detection in live cells and show poor selectivity and sensitivity.

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Principle

AcroleinRED is based on phenylazide-acrolein click chemistry discovered by Drs. Katsunori Tanaka et al., RIKEN.

Phenylazide Specific Reaction with Acrolein

The phenylazide group reacts specifically and rapidly with acrolein to form the cyclic product 4-formyl-1,2,3-triazoline, which reacts with neighboring biomolecules nonspecifically to form covalent bonds.

Phenylazide Specific Reaction with Acrolein

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Principle of Acrolein Detection by AcroleinRED

There are two possible intracellular and extracellular pathways.

Principle of Acrolein Detection by AcroleinRED
  • ⓵ Intracellular Acrolein
    AcroleinRED is membrane-permeable, so it permeates the cell membrane and reacts with acrolein intracellularly to form 4-formyl-1,2,3-triazoline.
  • ⓶ Extracellular Acrolein Released from Cell
    Acrolein produced by lipid peroxidation of cell membranes are released to extracellular space. Acrolein quickly reacts with AcroleinRED outside cells, and the reaction product 4-formyl-1,2,3-triazoline incorporated into cells through the endocytosis pathway.

⓵, ⓶ In both pathways, the reaction product, 4-formyl-1,2,3-triazoline, reacts with biomolecules (e.g., proteins) randomly in the cells and stays inside the cell to stain the cell.
Since the reaction products of acrolein, the reagent and biomolecules are detected as fluorescent signals, AcroleinRED does not visualize the localization of acrolein. Please note that localization cannot be observed with this reagent.

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Features

  • AcroleinRED is a reagent that takes advantage of the specific and rapid reaction of phenyl azide with acrolein. By labeling acrolein produced by cells with TAMRA, acrolein can be visualized.
  • Rhodamine filter sets are available. (Ex/Em: 560 nm/ 585 nm)
  • No react with other unsaturated aldehydes (crotonaldehyde, trans-2-octenal, methacrolein) or styrene
  • No pre-treatment and easy procedure
  • High sensitivity than existing acrolein quantification methods (detection limit: 100 nM)
  • Semi-quantification of acrolein amount by fluorescence intensity

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Application

  • Semi-quantification of acrolein production at steady state
  • Semi-quantification of effects in acrolein production by external stimuli (e.g., drug treatment)

AcroleinRED cannot be applied into localization study of intracellular acrolein.

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How to Use

AcroleinRED handling procedure

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Application Examples

Selectivity of Phenylazide to Acrolein

Detection Reagent

phenylazide    

phenylazide
Target

acrolein

acrolein

methacrolein

methacrolein

crotonaldehyde

crotonaldehyde

trans-2-octenal

trans-2-octenal

styrene

styrene
Reaction 47% <1% <1% <1% <1%

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Observation of Oxidative Stress-induced Acrolein Production

HUVECs were pretreated with 0-1,000 μM Hydrogen Peroxide (H2O2) for 2 hours and subsequently treated with 10 μM AcroleinRED for 30 min, then observed under live cell condition. In the absence of H2O2, the acrolein endogenously produced by HUVECs was observed. Intracellular TAMRA signals were increased in H2O2 dose-dependent manner compared with the endogenous acrolein level.

Acrolein Increase in HUVECs

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Observation of Reactive Oxygen Species (ROS)-induced Acrolein Production

HUVECs were treated with 25 μM Menadione, an inducer of reactive oxygen species (ROS), for 0, 10, 30, 60 min and subsequently co-stained with ROS detection dye and AcroleinRED. By the addition of Menadione, ROS were immediately increased, however, the acrolein level increased belatedly.

Photo comparing acrolein production by ROS-induced reagents
Chart comparing acrolein production by ROS-induced reagents

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Comparison of Acrolein-Production Levels of Various Cell lines by AcroleinRED

Relative levels of acrolein production were compared in three normal human cells and eight cancer human cell lines using AcroleinRED. Red fluorescent signals of cancer cells were much higher than that of normal cells. Furthermore, AcroleinRED revealed that acrolein-production levels of eight cancer cell lines are significantly different from each other

Cell lines by AcroleinRED
Chart_Cell lines by AcroleinRED

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Visualization of Tumors From Breast Gland Tissues

Acroleins in Tissues

Surgical tissues derived from breast cancer patients (IDC; invasive ductal carcinoma, DCIS; ductal carcinoma in situ) or a healthy control (NBG; normal breast gland) were immediately incubated with AcroleinRED (20 μM)/Hoechst mixed solution under non-fixed condition for 5 min. After washing tissues, double-stained tissues were observed by fluorescent microscopy. AcroleinRED visualized tumor region of patient-derived breast gland (IDC, DCIS), but not derived from healthy control (NBG). Thus, it is possible to stain extracted tissue in an unfixed state with this reagent


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Original Paper

  • A. R. Pradipta, M. Taichi, I. Nakase, E. Saigitbatalova, A. Kurbangalieva, S. Kitazume, N. Taniguchi, K. Tanaka, ACS Sens., 1, 623~632 (2016).
    Uncatalyzed Click Reaction between Phenyl Azides and Acrolein: 4-Formyl-1,2,3-Triazolines as “Clicked” Markers for Visualizations of Extracellular Acrolein Released from Oxidatively Stressed Cells.
  • T. Tanei, A. R. Pradipta, K. Morimoto, M. Fujii, M. Arata, A. Ito, M. Yoshida, E. Saigitbatalova, A. Kurbangalieva, J. Ikeda, E. Morii, S. Noguchi, and K. Tanaka, Adv. Sci., 1801479 (2018)
    Cascade reaction in human live tissue allows clinically applicable diagnosis of breast cancer morphology. [PMID:30693189]

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Product Information

[Date : May 30 2024 00:08]

Detail Product Name Product Code Supplier Size Price
AcroleinRED, Cell-based Acrolein Detection Reagent
DatasheetThis may not be the latest data sheet.
FDV-0022 FNAFunakoshi Co.,Ltd. 0.5 mg $380

Description Our AcroleinRED is the world first cell-based acrolein-detection reagent in live cells condition without any pre-treatment and cell lysis. This reagent is based on novel phenylazide-acrolein click chemistry discovered by Drs. Katsunori Tanaka and Ambara Pradipta (Ref.1). AcroleinRED specifically react with either extracellular acrolein conjugate released from cell surface lipids or intracellular acrolein generated via enzymatic pathway and label acrolein with TAMRA fluorophore.
Storage -20°C CAS
Link

Lipid Peroxidation Detection Tools for Cancer Research

[Date : May 30 2024 00:08]

AcroleinRED, Cell-based Acrolein Detection Reagent


  • Product Code: FDV-0022
  • Supplier: FNA
  • Size: 0.5mg
  • Price: $380

Description Our AcroleinRED is the world first cell-based acrolein-detection reagent in live cells condition without any pre-treatment and cell lysis. This reagent is based on novel phenylazide-acrolein click chemistry discovered by Drs. Katsunori Tanaka and Ambara Pradipta (Ref.1). AcroleinRED specifically react with either extracellular acrolein conjugate released from cell surface lipids or intracellular acrolein generated via enzymatic pathway and label acrolein with TAMRA fluorophore.
Storage -20°C CAS
Link

Lipid Peroxidation Detection Tools for Cancer Research

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CONTACT

export@funakoshi.co.jp

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