NO WASH STEPS
EASY SINGLE-STEP ASSAY
FATTY ACID UPTAKE AND TRANSPORTERS
Fatty acids are utilized for diverse cellular processes including mitochondria oxidation, membrane synthesis and energy storage. Pathologically increased intracellular fatty acid concentrations can cause cellular apoptosis and have been linked to insulin desensitization, Type 2 diabetes, obesity and cardiovascular disease. All of these intracellular processes are dependent upon fatty acids traversing the plasma membrane to get into the cell. Therefore, understanding the Fatty Acid Transport Proteins (FATP) that regulate this process is of great importance in biomedical research and drug discovery.
FATPs are part of an evolutionarily conserved family of proteins of which six FATP family members are expressed in the human genome. While all six human FATPs are similar in function, they differ dramatically in their expression in various tissue and organs. The differences in FATP expression pattern therefore make it possible to selectively target the uptake of fatty acids into specific organs by using compounds that can discriminate between the different FATP family members.
The development of fatty acid uptake regulators as potential drugs for metabolic diseases requires robust assays for the quantitation of fatty acid uptake. Conventional protocols utilizing radioactivity often require cell lysis and processing at very low temperature, making them expensive, slow and not suitable for high throughput screening. Fluorescence-based protocols generally require the use of low throughput Fluorescence Activated Cell Sorter (FACS) instrumentation, or require cell washing, which can severely com promise the integrity of fragile adipocyte cells.
The QBT Fatty Acid Uptake Assay is a single-step homogeneous fluorescent assay that provides real-time uptake kinetics and is ideally suited for high throughput screening applications in both 96-well and 384-well formats. The kit employs a BODIPY-dodecanoic acid fluorescent fatty acid analog coupled with Molecular Devices proprietary quench technology. The BODIPY label provides an ideal long chain fatty acid analog that behaves much like natural fatty acids: it becomes activated by acyl-CoA attachment; is incorporated into di-and triglycerides; and accumulates in intracellular lipid droplets. In addition, the BODIPY analog is a known substrate for fatty acid transporters since its uptake by adipocytes can be competed by non-labeled fatty acids. The elimination of radioactive compounds results in easier reagent handling, reduced disposal costs and eliminates safety risks associated with radiolabel assays.
QBT Assay Kits from Molecular Devices employ a quenching dye to reduce background fluorescence and improve the signal-to-noise ratio. The patented quench technology is offered to drug discovery and life science researchers exclusively by Molecular Devices, through the purchase of QBT Assay Kits.
OBSERVE TRUE BIOLOGICAL ACTIVITY
Consistent with the observation that FATPs are only expressed by differentiated cells, 3T3 L1 adipocytes take up the BODIPY analog and accumulate it in lipid droplets. In contrast, undifferentiated 3T3L1 fibroblasts do not transport or store the dye. (See Figure 1.) Known activators and inhibitors of fatty acid uptake can be seen to stimulate or inhibit uptake in adipocytes in a dose-dependent manner, but show no effect on uptake in undifferentiated fibroblasts. (See Figures 2 and 3.)
RAPID AS SAY DEVELOPMENT
The streamlined homogeneous format makes the QBT Fatty Acid Uptake Assay Kit less labor intensive to run. With the single step mix-andread procedure, the cells can be incubated with the reagents and are stable for up to several hours. Rapid analysis of the cells can be followed with the detection on any bottom-read fluorescence plate reader, including Molecular Devices Gemini EM, SpectraMax M5, Analyst, FlexStation and FLIPR microplate readers.
QBT Fatty Acid Uptake Assay Kit (Explorer) Part Number: R8132
(10) 1-plate reagent vials
Provides sufficient reagent for 10 plates (96- or 384-well)
QBT Fatty Acid Uptake Assay Kit (Bulk) Part Number: R8133
(10) 5-plate reagent vials
Provides sufficient reagent for 50 plates (96- or 384-well)
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Analyst, FlexStation, FLIPR and SpectraMax are registered trademarks and QBT is a trademark of Molecular Devices Corporation.
BODIPY is a registered trademark of Molecular Probes, Inc.
Quenching technology is covered under U.S. Patent 6,420,183 and EPO Patent 0 906 572.