art-ffa-ss-luc-fatty-acid-uptake-probe

Fatty acid uptake probe

 290.00 1855.00

Highly sensitive (0.01 mg/mouse) fatty acid uptake probe.

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Introduction

Strategy for bioluminescence imaging of fatty acid uptake
Figure 1. Strategy for bioluminescence imaging of fatty acid uptake. The FFA probe is taken up by a transporter-mediated process (Fatty-acid transport protein, FATP). The disulfide bond is reduced by intracellular glutathione, and the resultant thiol undergoes cyclization releasing free luciferin. Luciferase-catalyzed conversion of luciferin to oxyluciferin is accompanied by production of a photon of light, which is detected using a charge-coupled device (CCD) camera1. Reproduced from ACS Chem. Biol., 2012, 7(11), 1884-1891. Copyright 2012 American Chemical Society.

Fatty Acids are carboxylic acids with a long saturated or unsaturated aliphatic tail. Fatty acids are very important sources of energy because they produce a large amount of ATP when metabolized. To enable noninvasive, real-time and spatiotemporal quantitative imaging of fatty acid fluxes in animals, we developed a bioactivatable molecular imaging probe FFA-SS-Luc based on bioluminescence1.

The bioluminescent probe D-luciferin is attached to a long fatty acid chain via a disulfide bridge that is stable in the extracellular matrix but rapidly reduces by intracellular glutathione after cellular uptake. Alkylation of Luciferin on the phenolic oxygen prohibit the generation of light because they are not a substrate for Luciferase.2 After FFA-SS-Luc cell internalization, glutathione reduces the disulfide bridge and D-luciferin is generated instantaneous through cyclization of the linker. Free luciferin is then converted by luciferase to oxyluciferin and produce a photon of light that can be detected and quantify by CCD camera (Figure 1).3

The FFA-SS-Luc probe can be used for in vitro as well as in vivo to sensitively detect fatty acid uptake by bioluminescence.

Fatty-acid transporter inhibitor (FFAi) screening assay

The FFAi screening assay is a non-radioactive assay that can be used for screening of potential fatty-acid transporter inhibitors in vitro and in vivo. These robust assay uses bioluminescence as readout in high throughput. Below is the validation of the FFA-SS-Luc probe in vitro where the probe kinetic uptake has been monitored in two different cell lines. The probe show high specificity for Adipocytes (high FATP expression) compared to fibroblasts (Low expression of FATP).

Figure 2. Image overlay of a photographic image and bioluminescence from 96-well plates containing equal number of 3T3-luc Fibroblasts (upper row) or adipocytes (lower row) incubated with the indicated concentrations of FFA- SS-luc for 10, 20, and 30 min. Scale min: 1.03×103 and max: 2.14×104 p/s (photons/second)1. Reproduced from ACS Chem. Biol., 2012, 7(11), 1884-1891. Copyright 2012 American Chemical Society

FFA-SS-Luc for in vivo imaging

The FFA-SS-Luc probe can be used to localize and quantitate in real time lipid fluxes in living animals with or without treatment. Below is represented a live-bioluminescence imaging of FFA-SS-Luc uptake in full-luciferase expressing mouse overtime.

Figure 3. Ventral luminescent/photographic overlay sequence of animals following a gavage with 100 μL FFA-SS-luc (20 μM) in cremophor1. Reproduced from ACS Chem. Biol., 2012, 7(11), 1884-1891. Copyright 2012 American Chemical Society

FFA-SS-luc probe enables the interrogation of FFA uptake in real time in vivo by bioluminescence imaging. Bioluminescence imaging allows spatiotemporal and quantitative measure of fatty acids uptake in vivo. This probe can also be used for drug screening to limit fatty acid uptake and can benefit understanding fundamental biological processes.

Material Amount Storage Stability

FFA-SS-luc

Fatty acid uptake probe

0.5 mg
  • -20° C
  • Desiccate
  • Protect from light
When stored as directed, reactive probes are stable for at least 3 months
1 mg
5 mg

Immediately before use, dissolve the FFA-SS-Luc probe in DMSO to give a 10 mM stock solution (e.g. for 1 mg of FFA-SS-Luc add 146 uL of DMSO). Stock solution in DMSO can be kept at -20C for few weeks.

Cell-based assay

Cells of interest are seeded into black-wall/clear-bottom-96-well plates. FFA-SS-Luc stock solution is diluted to appropriate concentration (e.g. 2-100 μM) in PBS 7.4 supplemented with 0.1% fatty-acid free BSA (w/v) (e.g. A3803 Sigma). 100 μL of FFA-SS-luc or control compound is then added to the cells and bioluminescent readout is immediately performed with a 5-min exposure time for 60 min1.

In Vivo assay

Gavage of FFA-SS-luc.

FFA-SS-Luc stock solution is diluted to 300 μM in a vehicle of 1:1 poly(ethylene glycol) (PEG 400) and propylene glycol (PG). 50 μL of the 300 μM FFA-SS-luc solution was given to anesthetized mice by gavage. After 5 min postgavage mice were reanesthetized and luminescent images were acquired with a 5-min exposure for 60 min1.

 

Intravenous Injection of FFA-SS-luc

FFA-SS-Luc stock solution is diluted to 20 μM with PBS 7.4 supplemented with 0.1% fatty-acid free BSA (w/v). 100 μL of 20 μM FFA-SS-luc or control is then administrated by tail vein injections. Mice were immediately anesthetized and images with a 1-min exposure1.

 

Intraperitoneal Injection of FFA-SS-luc and FFA-S-luc.

FFA-SS-Luc stock solution is diluted to 200 μM with PBS 7.4 supplemented with fatty-acid free 0.1% BSA (w/v). 100 μL of 200 μM FFA-SS-luc or control is then administered to anesthetized mice by IP injection. Mice were immediately images with a 3-min exposure for 30 min1.

 

Note: Solutions of FFA-SS-luc probe should be discarded after use.

Once reconstituted, this reactive probe solution is somewhat unstable, stability test in solution showed non-specific hydrolysis with a half-life of 432 min at 37C.

Figure 4. Stability of FFA-SS-Luc in PBS 7.4 + 0.1 % BSA

  1. Henkin, A. H., Cohen, A. S., Dubikovskaya, E. A., Park, H. M., Nikitin, G. F., Auzias, M. G., Kazantis, M., Bertozzi, C. R., Stahl, A. (2012) Real-Time Noninvasive Imaging of Fatty Acid Uptake in Vivo. ACS Chem. Biol. 7(11), 1884-1891
  2. Denburg, J. L., Lee, R. T., and McElroy, W. D. (1969) Substrate- binding properties of firefly luciferase. Arch. Biochem. Biophys. 134, 381-394.
  3. Jenkins, D. E., Oei, Y., Hornig, Y. S., Yu, S., Dusich, J., Purchio, T., and Contag, P. R. (2003) Bioluminescent imaging (BLI) to improve and refine traditional murine models of tumor growth and metastasis. Clin. Exp. Metastasis 20, 733-744.

Fatty acid uptake probe

  • Cleavable luciferin technology to monitor and quantify fatty acid uptake in vivo.
  • Use in cells and animals expressing luciferase
  • Available today at a very reasonable price
Real-time noninvasive imaging of fatty acid uptake in vivo. ACS Chem Biol. 2012, 7(11), pp. 1884-91
Fatty ACID
Reproduced from ACS Chem. Biol., 2012, 7(11), 1884-1891. Copyright 2012 American Chemical Society.
Ventral luminescent
Ventral luminescent / photographic overlay comparing the BLI of FFA-SS-luc (right) and the control FFA-S-luc (left) 20 min postgavage.
Color

Light yellow powder

Bioluminescent Label

Luciferin

Product Size

1 kit

Detection Method

Bioluminescense

Excitation Class

Visible

Molecular Weight

684.95 g.mol-1

Formula

C31H44N2O7S4

Shipping Condition

Dry Ice

Regulator Statement

For Research Use Only. Not for use in diagnostic procedures.

Quantity

1 mg, 5 mg, 10 mg

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