QBT Fatty Acid Uptake Assay Kit
Frequently Asked Questions
Why is fatty acid metabolism important?
Defects in fatty acid metabolism have been linked to several pathological states, including
Type 2 diabetes and obesity which are reaching epidemic proportions. It is estimated that 20
million Americans have diabetes and 400 million are obese.
Recognizing this trend, nearly all major pharmaceutical companies are involved in this area
of research. Past attempts to modulate fat absorption by inhibiting lipases have resulted in
undesirable side effects (e.g., anal leakage). Therefore, selective inhibition of fatty acid
uptake would bypass these side effects and could improve insulin signaling in skeletal
muscle, insulin sensing in liver, and reduce fat absorption in the intestine, leading to reduced
pathology.
How is fatty acid uptake typically measured?
Most commonly, fatty acid uptake is measured using radiolabeled fatty acids. The procedure
involves a wash step, which is tough on fragile adipocytes, an on-ice incubation, cell lysis
and reading in a scintillation counter. This is time and labor intensive. Alternatively, and far
less common, uptake of fluorescently-labeled fatty acids can be measured on a cell sorter,
following washing, centrifugation and fixation.
What are the advantages of the new QBT kit over current methods?
The QBT kit is a no-wash, homogenous, reproducible assay. It does not require radioisotope
usage (and the inherent safety and disposal concerns), cell lysis, on-ice incubations or highly
specialized and expensive equipment.
The QBT assay incorporates MDC's proprietary quench technology, therefore there are no
wash steps involved which would disrupt the fragile adipocytes. It is a simple fluorescent
assay and can be read on any bottom-read type fluorometer.
How will this assay be used?
The QBT assay is ideal for high-throughput screening. However, it will also enable basic
research of fatty acid metabolism. For example, when using this assay Cambridge
Biosciences was able to demonstrate Leptin inhibition of fatty acid uptake in vitro for the first
time.
What type of instrument is required for this assay?
Any bottom-read fluorescent plate reader can be used for this assay. Ideal MDC instruments
for this assay include the Analyst®, Gemini™ and SpectraMax® M5. The FLIPR® and
FlexStation® can also be used with this assay.
What is the size of a typical fatty acid uptake screen?
Conventional fatty acid uptake assays are not amenable to high-throughput screening, so
there is no typical screen. However, a small screen has been run with the QBT Fatty Acid
Uptake Kit. Now that the homogeneous QBT Fatty Acid Assay is available, we anticipate
that large pharma will run much higher throughput screens using this assay.
Is the assay temperature dependent?
We have not detected significant temperature dependence and regularly perform the assay at
room temperature. However, in some cases performance may be improved by running the
assay at 37 °C.
Is the assay a kinetic or end-point read?
Fatty acid uptake is a slow process. Although there may be some kinetic differences in the
first 5-10 minutes of the assay, it is typically read as a single end point after 30-60 min.
What cells can be used in this assay?
Differentiated 3T3 L1 adipocytes are the preferred cell type for the assay. Differentiated
adipocytes cannot be shipped, so the differentiation, which takes approximately 3 weeks,
must be done by the researcher. The assay can also be performed in human adipocytes
(available from Cell Applications, Inc.) or a cell line (e.g., CHO) that has been transfected
with and expresses a fatty acid transporter (FATP).
What can go wrong with this assay?
The assay is so straightforward that little can go wrong with the assay itself. If, however, a
customer experiences a poor assay response, we have found that it is usually related to the
cells rather than the assay performance. For example, 3T3-L1 adipocytes need to be
differentiated from their fibroblastic form. Fibroblasts do not have expressed FATP
transporters, thus no signal will be observed. In fact, undifferentiated 3T3 cells can
sometimes serve as a negative control. If the cells are not fully differentiated, a poor signal
will be obtained (low fluorescence). Poor differentiation can be observed under a
microscope. If the customer is having 3T3 differentiation problems, it is recommended that
they refer to the differentiation protocol in the Product Insert.
As in GPCR assays, it is important to verify that, if cells are transfected with FATP
transporters, they are indeed expressing them.
How do I order the QBT Fatty Acid Assay?
Please visit the Molecular Devices website (www.moleculardevices.com) or see your local
sales representative to place an order or request more information.
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