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FAOBlue (Fatty Acid Oxidation Detection Reagent)

Product#: FNK-FDV-0033
$418.60
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Funakoshi · Cell Imaging Probes

FAOBlue™ (Fatty Acid Oxidation Detection Reagent)

Directly detects fatty acid β-oxidation (FAO) activity in live cells; FAO releases a coumarin dye that fluoresces blue (Ex 405 nm / Em 460 nm).
For Research Use Only.

Funakoshi Co., Ltd. · Distributed by Diagnocine
Cat No.
FNK-FDV-0033
UNSPSC Code
12352206
Size
0.2 mg + 500 mL HBS
Storage
Reagent: -20°C (long-term) / HBS 2-8°C

Tip — The Best Match Bundle Products for Lipid Metabolism Research: LipiDye II (lipid droplet staining) with FAOBlue (Fatty Acid Oxidation Detection Reagent). View the bundle

Shipping: Room temperature. The reagent ships as a stable powder.

Protocol Application Data Webinar Publications References

Overview

FAOBlue is a fluorescent detection system for studying the catabolic pathway of energy production. It visualizes fatty acid β-oxidation (FAO), the common pathway of fatty acid degradation, as blue fluorescence. FAO activity, which was previously difficult to measure, can be measured by fluorescence imaging simply by adding the dye to cells.

FAOBlue fatty acid oxidation detection in live cells - animation

Background: Fatty Acid β-Oxidation

Fatty acids (FAs) are the basic building blocks of a wide variety of lipids, are essential components of cells, and are one of the primary sources of energy. The major pathway for FA degradation is mitochondrial FA β-oxidation (FAO), a key metabolic pathway for energy homeostasis in organs such as the liver, heart and skeletal muscle.

FAO is a complex biochemical process involving many enzymes. First, FAs are converted to acyl-CoA by the acyl-CoA synthetase family. Second, acyl-CoA enters mitochondria via the carnitine shuttle. In the mitochondrial matrix, acyl-CoA (Cn) is converted to acyl-CoA (Cn-2) and acetyl-CoA by four stepwise reactions:

1 · Dehydrogenation
Acyl-CoA is oxidized to enoyl-CoA by acyl-CoA dehydrogenases
2 · Hydration
Enoyl-CoA is hydrated to 3-hydroxyacyl-CoA by crotonase
3 · Oxidation
3-Hydroxyacyl-CoA to 3-ketoacyl-CoA
4 · Thiolysis
3-Ketoacyl-CoA to acyl-CoA (Cn-2) and acetyl-CoA

Acetyl-CoA is further converted to ATP. The resulting acyl-CoA (Cn-2) enters another FAO cycle to produce acyl-CoA (Cn-4).

Abnormal FAO is involved in diseases such as obesity and fatty liver disease (MASLD). Although measuring FAO activity in diseased cells is important, available methods are limited because of the complexity of the pathway. Only a few indirect methods, such as radioisotope-labeled fatty acids or oxygen-consumption measurement, are commonly used.

Detection Principle

FAOBlue structure and detection principle
POINT

FAOBlue is the world-first reagent for directly measuring FAO activity in living cells. Before FAO metabolism it is non-fluorescent under 405 nm excitation; the coumarin released by FAO cycles fluoresces strongly blue under 405 nm excitation.

FAOBlue is a coumarin dye carrying a nonanoic acid (C9) protected by an acetoxymethyl ester.

Step 1 · Uptake
FAOBlue enters cells by direct penetration of the cell membrane; intracellular esterases hydrolyze the acetoxymethyl ester
Step 2 · Activation
The free-FA form is converted to acyl-CoA and incorporated into the FAO pathway
Step 3 · Three FAO cycles
The acyl-CoA form is degraded to a non-fluorescent coumarin carrying a propionic acid (C3)
Step 4 · Release
After the 4th FAO degradation, the coumarin dye is released and diffuses through the whole cell

The FAOBlue-based FAO assay measures FAO activity with an easy procedure. Inhibition of the carnitine shuttle by etomoxir greatly diminishes the fluorescence enhancement in living cells, which indicates that FAOBlue mainly detects mitochondrial FAO activity.

FAOBlue fluorescence enhancement and etomoxir inhibition FAOBlue metabolism by fatty acid beta-oxidation

Key Features & Applications

Direct live-cell readout

Measures FAO activity directly in living cells, not through radioisotopes or oxygen consumption.

Add-and-image workflow

Add the dye to cells, incubate, wash and image with 405 nm excitation.

Relative quantification

Relative quantification of FAO activity.

Drug evaluation

Evaluation of drug effect on FAO activity.

Components & Specifications

The following components are included with this product.

(a) FAOBlue Reagent
Size 0.2 mg
(0.2 mg ≈ 21–84 mL working solution (5–20 µM)
≈ 200–800 wells at 100 µL)
Molecular formula C24H31NO9
Molecular weight 477.51 g/mol
Solubility Soluble in DMSO
Storage -20°C (long-term storage)
(b) HEPES-Buffered Saline (HBS Buffer)
Size 500 mL
Sterile Filtered 0.1-micron membrane TWICE, 0.04-micron membrane ONCE
Storage 2-8°C, away from bright light
Component HEPES (at pH 7.4): 20 mM (4766.024 mg/L)
Sodium chloride (NaCl): 107 mM (6253.08 mg/L)
Potassium chloride (KCl): 6 mM (447.309 mg/L)
Magnesium sulfate (MgSO4): 1.2 mM (144.439 mg/L)
Calcium chloride (CaCl2): 2 mM (221.960 mg/L)

Optical Properties

Excitation
405 nm
Emission
460 nm
Imaging channel
Ex 405 nm / Em 430-480 nm
Shipping
Room temperature

Reconstitution & Storage

Reconstitution
Add 100% DMSO to the vial to prepare a 1-10 mM stock solution.
Powder
Store at -20°C, protected from light.
Stock solution
Aliquot and store at -20°C, protected from light.
Handling
Avoid repeated freeze-thaw cycles.

Experimental Guide

Absorbance and Fluorescence Spectra of FAOBlue and the Coumarin Derivative

Absorbance (left) and fluorescence (right) spectra of FAOBlue (blue line) and the coumarin-derivative dye released after FAO metabolism (red line) in PBS buffer (pH 7.4).

  • Absorbance spectra: The absorption peak of the coumarin-derivative dye is clearly shifted by FAO from the peak of FAOBlue.
  • Fluorescence spectra: When both compounds are excited at 405 nm, the coumarin-derivative dye shows strong blue fluorescence while FAOBlue emits little fluorescence.
NOTE: FAOBlue shows blue fluorescence (gray line; 370-450 nm) when excited at 300-380 nm (max 350 nm).
Absorbance and fluorescence spectra of FAOBlue and coumarin derivative

Imaging Guide

Confocal laser microscopy: Use the 405 nm laser of the microscope. A 405 nm laser detects only the fluorescent signal from the coumarin-derivative dye.

Epifluorescence microscopy — the excitation filter is critical. Commercial DAPI filters are not compatible with this reagent, because DAPI filters excite both FAOBlue and the coumarin-derivative dye. Excitation filters that pass 390-450 nm light are recommended.

Protocol: General Procedure

Step 1
Add FAOBlue (recommended final conc. 5-20 µM) to fresh HEPES-buffered saline (HBS).
NOTE: Serum- and phenol red-free medium can replace HBS.
Step 2
Remove the culture medium and wash the cells twice with HBS.
Step 3
Add the FAOBlue-containing HBS to the cells.
Step 4
Incubate the cells at 37°C for >30 min.
Step 5
Wash the cells with HBS.
Step 6
Observe live cells by blue fluorescence (Ex 405 nm / Em 430-480 nm).
FAOBlue general staining procedure

Bio-protocol: FAOBlue protocol

Application Data

Visualization of FAO Activity in Four Cancer Cell Lines

Four cancer cell lines (HepG2, LNCaP, HeLa and A549) were treated with FAOBlue in HBS buffer with or without pre-treatment with etomoxir (40 µM, 3 hours), a potent FAO inhibitor. After FAOBlue incubation, blue fluorescence (Ex 405 nm / Em 430-480 nm) was observed. All cell lines showed blue fluorescence in the cytosol, and etomoxir pre-treatment clearly decreased the fluorescence intensity, indicating that the blue fluorescence derived from FAO activity in the cells.

Cell line FAOBlue Incubation
HepG2 5 µM 30 min
LNCaP 20 µM 120 min
HeLa 20 µM 120 min
A549 5 µM 30 min
FAOBlue imaging of FAO activity in HepG2, LNCaP, HeLa and A549 cells with and without etomoxir

Perturbation of FAO Activity by Drugs

HepG2 cells were pre-incubated with 200 µM AICAR, an FAO activator via AMPK activation, for 3 hours, or with 200 µM ranolazine, a partial FAO inhibitor, for 12 hours. The cells were then incubated with 5 µM FAOBlue for 30 min. Compared with control cells, AICAR pre-treatment significantly increased blue fluorescence intensity, while ranolazine pre-treatment clearly decreased it. Ex 405 nm / Em 430-480 nm.

FAOBlue fluorescence in HepG2 cells treated with AICAR or ranolazine

Quantitative Analysis of Drug Effects on FAO Activity

ND630, an inhibitor of acetyl-CoA carboxylase, is considered a potential drug for MASLD. HepG2 cells were pre-incubated with various concentrations of ND630 for 4 hours, then treated with 5 µM FAOBlue for 30 min. Blue fluorescence intensity was quantified at each ND630 concentration. Ex 405 nm / Em 430-480 nm.

Quantification of FAOBlue fluorescence versus ND630 concentration in HepG2 cells

Analysis of FAO Activity in a MASH Model Mouse

MASH (formerly NASH) shows low FA metabolic activity. Control healthy mice and MASH model mice were orally administered 400 mg/kg bezafibrate, a therapeutic agent for MASH. After 4 weeks of administration, primary hepatocytes were isolated from control and MASH model mice and cultured in culture dishes, then treated with 5 µM FAOBlue for 30 min and imaged. Compared with control cells, hepatocytes from MASH model mice showed low FAO activity, and bezafibrate dramatically recovered their FAO activity. FAOBlue is a powerful tool for estimating drug effects and efficiency on FAO activity.

The detailed procedure of the mouse experiment is described in Ref. 1. Ex 405 nm / Em 430-480 nm.
FAOBlue imaging of primary hepatocytes from control and MASH model mice with bezafibrate

Webinar: FAOBlue (Fatty Acid Oxidation Detection Reagent)

Lipid Metabolism Research Tools

Lipid metabolism research products
FAOBlue fatty acid oxidation detection reagent overview
Fatty acid oxidation pathway and FAOBlue
BioTechniques

Featured in BioTechniques. An international life-science journal.

Research Applications

FAOBlue directly measures fatty acid oxidation (FAO) activity across diverse biological systems. Unlike indirect methods such as oxygen consumption or radioisotopes, it allows real-time, fluorescence-based visualization of mitochondrial β-oxidation.

Metabolic Research & Disease Models

Studies of metabolic dysfunction, particularly in the liver and heart.

  • MASLD/NAFLD: Yu et al. (2025) and Chen et al. (2023) used FAOBlue to show how protein interactions (Asprosin-FABP5) or exercise-induced pathways (Cdo1-AMPK) modulate fatty acid breakdown in liver steatosis.
  • Cardiac health: Tian et al. (2023) showed that VDAC1 overexpression alleviates cardiac fibrosis by regulating fatty acid metabolism.
  • Kidney injury: Yuan et al. (2025) found that inhibiting FAO via PRMT1-mediated methylation exacerbates crystal-induced kidney injury.
Immunology & Immunotherapy

Assessment of the metabolic state of immune cells, which often shift their energy source during activation.

Oncology & Cell Survival

Cancer cells often rewire their metabolism to survive harsh conditions.

  • Survival mechanisms: Xiong et al. (2024) used FAOBlue in breast cancer cells under glucose starvation.
  • Therapeutic response: Nandi et al. (2024) showed that targeting FAO enhances response to HER2-targeted therapy.
Developmental & Specialized Biology

Applications in biological timing and stress.

  • Embryonic dormancy: Lipid metabolism maintains mammalian embryos in diapause (Weijden et al., 2024).
  • Cellular senescence: Yamauchi et al. (2024) found that mitochondrial fatty acid oxidation drives senescence.
  • Neurogenesis: Jia et al. (2026) showed that sevoflurane impairs neurogenesis and cognitive function by inhibiting FAO in neural stem/progenitor cells.

Featured Publication

The short-chain fatty acid acetate modulates epithelial-to-mesenchymal transition

Lyu et al., Molecular Biology of the Cell (2022) · DOI: 10.1091/mbc.E22-02-0066

View article

Ordering Information

FAOBlue (Fatty Acid Oxidation Detection Reagent)
Product Cat No. Size Order
FAOBlue (Fatty Acid Oxidation Detection Reagent) FNK-FDV-0033 0.2 mg + 500 mL HBS Current page

References

This product was commercialized and is sold by Funakoshi Co., Ltd. based on the research of Professor Akio Ojida (chemical biology), Graduate School of Pharmaceutical Sciences, Kyushu University. Research use only; not for diagnostic or therapeutic use.

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Paper citation material — updated Oct 8, 2026.

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