Cultured Human Adipocyte Lipolysis Assay Kit
Non-Esterified Fatty Acids Detection
100 point assay kit
Cat# LIP-2; LIP-2-NC
INSTRUCTION MANUALZBM0010.03

STORAGE CONDITIONSHuman Adipocytes
All orders are delivered via Federal Express Priority courier at room temperature.

All orders must be processed immediately upon arrival.
NOTE:
Domestic customers: Assay must be performed 5-7 days AFTER receipt.
International customers: Assay must be performed 3-5 days AFTER receipt
Reagents & Buffers: 4°C
Vehicle & Controls: -20°C
Assay plate A (96-well) cultured human adipocytes: 37°C
For in vitro Use Only
LIMITED PRODUCT WARRANTYThis warranty limits our liability to replacement of this product. No other warranties of any kind, expressed or implied, including
without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Zen-Bio, Inc. Zen-Bio, Inc.
shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or theinability to use this product.

ORDERING INFORMATION AND TECHNICAL SERVICESZen-Bio, Inc.
3200 Chapel Hill-Nelson Blvd., Suite 104
PO Box 13888
Research Triangle Park, NC 27709
Telephone
Facsimile (FAX)
Toll Free(919) 547-0692(919) 547-0693
1-866-ADIPOSE

Electronic mail (e-mail)information@zen-bio.com

World Wide Web

http://www.zenbio.com

Rev 8/15/2008

Page 1 of 8(866)-234-7673

INTRODUCTION
Lipolysis plays a central role in the regulation of energy balance. Lipolysis is the process in which
triglycerides are hydrolyzed into glycerol and free fatty acids. This process releases free fatty acids (FFA) into thebloodstream where they may be either re-esterified by the adipocyte or travel to other tissues and exert othereffects throughout the body. Elevated adipocyte lipolysis has been observed in obese and diabetic individuals(Arner 1996). Alterations in lipolytic capacity have also been implicated in the susceptibility to obesity of AfricanAmerican individuals versus their Caucasian cohorts (Danadian et al. 2001).
The sympathetic nervous system plays a key role in the regulation of lipid mobilization. The main lipolytic
pathway involves beta-agonists ( -agonists), which activate -adrenergic receptors via the intracellular Gs proteinsin adipocytes. This leads to the activation of adenylate cyclase (AC), which then increases cyclic AMP (cAMP)levels. Elevated cAMP acts as a second messenger to activate hormone sensitive lipase (HSL). HSL, the ratelimiting enzyme regulating adipocyte lipolysis, then catalyzes the hydrolysis of triglycerides and results in therelease of glycerol and FFA (increased lipolysis). Phosphodiesterases (PDE) are enzymes that hydrolyze cAMPto 5’-AMP (5 prime adenosine monophosphate). This action results in a decrease in lipolysis. PDE inhibitors
increase intracellular cAMP levels. 3-isobutyl-1-methylxanthine (IBMX), a non-specific inhibitor of cAMP
phosphodiesterases (PDE), is used as the positive control if your test compounds are suspected PDE inhibitors.
Isoproterenol, a non-specific -adrenergic agonist is used as the positive control if your test compounds affectlipolysis via -adrenergic receptors.
This lipolysis assay kit provides the tool to study chemical compounds that may influence lipolysis incultured human adipocytes.
Figure 1. Overview of adipocyte lipolysis
EPINEPHRINE

1,NOREPINEPHRINE

2,3 AR

ACGs

IRPDE

ATPcAMPP

5’-AMP
PKATG

Per

HSL

FFA + glycerol

FFA + glycerol

bloodstream

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ABBREVIATIONS:
AC
adenylate cyclaseARadrenergic receptors
Gs
G protein coupled receptor
FFA
free fatty acids
PKA
protein kinase
AMP adenosine monophosphate
ATPadenosine triphosphateIRinsulin receptor
PDEphosphodiesteraseTG
triglyceridePRINCIPLE OF THE ASSAY
Assessment of lipolytic activity is through a coupled reaction to measure non-Esterified fatty acids (NEFA)
released by adipocytes. The initial step, carried out by acyl-CoA synthetase (ACS), produces fatty acyl-CoA thiol
esters from the NEFA, ATP, Mg, and CoA in the reaction. The acyl-CoA derivatives react with oxygen in theACS
presence of acyl-CoA oxidase (ACOD) toHCOOH + ATP + CoA
Acyl-CoA + AMP + PPi
produce hydrogen peroxide.
Hydrogen(NEFA)
peroxide in the presence of peroxidase (POD)
ACOD
allows the oxidative condensation of 3Acyl-CoA + O2
2,3-trans-Enoyl-CoA + H2O2
methyl-N-ethyl-N-( -hydroxyethyl)-aniline with4-aminoantipyrine which forms a purple
CHNH
product that absorbs light at 550nm. ThisNNC H OH
allows the concentration of NEFA to beNOCHPODNN2H O++ONN
determined from the optical density measured+ 4H O
C H OH
at 540 - 550nm.
222524252

422

ITEMS INCLUDED IN THE KITITEM

DESCRIPTION

Adipocytes, Plate
A
Assay Plate
LIP-2/3 Assay
Buffer
Wash Buffer
VehicleCultured human subcutaneous adipocytes (LIP-2only)
96-well assay plate, blank (for samples & standards
100 mlPositive control

Isoproterenol, 10 mM in DMSO. Dilute to 1 M in
Assay Buffer before use! (i.e.1 l in 10 ml AssayBuffer)
1mM Stock. See page 5 for standard curve
preparation

FFA Standard

50 ml
0.1% DMSO in LIP-2/3 Assay Buffer

FFA Diluent A

CapColor---UNIT

QTY

STORAGE

PLATE

137°C

PLATE

21----4°C

114°C
-20°C

-----

BOTTLE---BOTTLEPURPLE

1 ml /VIAL

BLUE

10 l /
VIAL

1-20°CAMBER

100 l /
VIAL

14°C

LABEL

10.5ML

14°C

LABEL PINK

5.5ML

LABEL

BOTTLE

114°C
4°C

LABEL PINKBOTTLE

14°C

CLEAR

EACH

2-----YELLOW

FFA Diluent B
FFA Reagent A

Reconstitute using 10.5 ml FFA Diluent A. Discard
remainder after 10 days
FFA Reagent B
Reconstitute using 5.5 ml FFA Diluent B. Discard
remainder after 10 days
Tray
For multi-channel pipetters, clear polyvinyl
Other equipment/reagents required but not provided with the kit:
Multi-channel Pipet , single channel pipet and pipet tips
Plate reader with a filter of 540 nm
Incubator at 37oC
Large gauge needle
Tubes for dilution of standards

Rev 8/15/2008Page 3 of 8YELLOWASSAY PROCEDURE
1. Preadipocytes are plated in 96 well plates and allowed to differentiate under standard Zen-Bio

differentiation conditions for 1 week. Upon arrival, remove 150 l of the shipping medium fromeach well and discard. Place the plate (Plate A) in your incubator for 5-7 days (3-5 days forinternational customers) to allow the cells to recover from the stress of shipping. To ensure
optimal performance, DO NOT feed the cells fresh medium during this time. Please observe thecells under a microscope prior to performing the assay [see the photograph in the Certificate ofAnalysis for the lot # of Plate A].
2. Make your stock solution using whatever vehicle is appropriate for your test compounds. Dilute

your stock solutions to their final concentration in LIP-2/3 Assay Buffer (100 ml is available).
NOTE: if desired, maintain a constant concentration of solvent by preparing all compound
dilutions in the highest concentration of that solvent. Dilute your controls in assay buffer. Prepare
all vehicles as appropriate for your compounds, 0.1% DMSO has been included as the vehicle forthe positive controls. Include the Assay Buffer alone as a vehicle control. PLEASE NOTE: ZENBIO DOES NOT RECOMMEND THE USE OF SOLVENTS AT CONCENTRATIONS ABOVE 1%.
3. Remove 120 l medium from each well. Gently add 200 l Wash Buffer to all wells. Remove 200

l of the media and Wash Buffer from each well and replace with another 200 l Wash Buffer.
4. Remove all the media and Wash Buffer from the cells from triplicate wells. Treat the cells with 75

l of the test compounds resuspended in Assay Buffer three (3) wells at a time. Treat with thediluted Isoproterenol as positive control. Use the Assay Buffer alone as one of the vehicle
controls. Please be sure to include both the vehicle provided in the kit and your vehicle (if yourtest compounds are not dissolved in DMSO). The assay should be performed in triplicate.
5. Incubate the plates at 37oC-humidified incubator for 3 hours (for time course experiments thelongest time point recommended is 5 hours).

Note: Treatment times longer than 3 hours will

result in significant fatty acid reutilization by the adipocytes and may decrease signal relative tototal lipolysis activity.
6. Prepare the standard curve using the STANDARD SOLUTION as follows:

Briefly spin down the contents of the free fatty acid standard tube before reconstitution.
Standards are: 0, 1.4, 4.1, 12.3, 37, 111, and 333 M fatty acid. Prepare as follows:

Rev 8/15/2008

Page 4 of 8

The kit standard solution is the 1.0 mM standard. Pipette 60 l of Dilution Buffer into 6tubes (not provided). Pipette 30 l of the FFA Standard Stock into a tube labeled 333 µM.
Prepare a dilution series as depicted below. Mix each new dilution thoroughly before
proceeding to the next. The Dilution Buffer alone serves as the zero standard.

30 l30 l 30 l 30 l 30 l

30 l

StdFFA
Std333
M

111
M37
M

12.3
M

4.1
M

1.4
M

7. Add 10.5ml FFA Diluent A to the FFA Reagent A bottle and gently invert. DO NOT VORTEX!

Store any remaining solution at 2-8 C; it is stable for 10 days after reconstitution refrigerated (28 C).
8. At the end of the incubation, 50

l of the conditioned media is removed and transferred to thecorresponding well of a blank plate for assessment of non-esterified fatty acids. [This is mosteasily accomplished using a multi-channel pipet.] Add 50 l of each standard to empty wells.
9. Add the reconstituted FFA Reagent A to one of the disposable trays provided in the kit. Add 100

l of FFA Reagent A to each well. Gently shake the plate to ensure mixing. Place in a 37 oC
incubator for 10 minutes.
10. Add 5.5 ml FFA Diluent B to the FFA Reagent bottle and gently invert. Store any remaining

solution at 2-8 C; it is stable for 10 days after reconstitution refrigerated (2-8 C).
11. Add the reconstituted FFA Reagent B to the other disposable tray provided in the kit. Add 50 l ofFFA Reagent B to each well. Gently shake the plate to ensure mixing. Place in a 37 oC incubatorfor 10 minutes.
12. Allow the plate to equilibrate to room temperature for 5 minutes. During this time, ensure that

there are no bubbles in the solution mixture. Use a large gauge needle or clean pipet tip to pop
any bubbles as this will result in inaccurate absorbance readings.
13. The optical density of each well is then measured at 540 nm.
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FATTY ACID STANDARD CURVE
Generate standard curve: see example below[DO NOT use this standard curve to generate your data. This is an example.]
Subtract the OD value of the 0 M standard from all OD values including the standard curve. Note: 1mM
standard is commonly omitted from analysis due to lack of linearity between 333 M and 1mM. Optionally, a 4parameter fit may be used to calculate standard curve.

OD
0.68
0.244
0.104
0.063
0.05
0.046
0.044

y = 0.0019x - 0.0045
R2 = 0.9995

OD - zero
0.636
0.2
0.06
0.019
0.006
0.002
0Standard Curve
0.75

0.50

O.D.

M std333
11137
12.3
4.1
1.4
00.25

0.00
0100200300

400

M standard

Data are expressed as M free fatty acids released.
OPTION: express data as Fold induction over appropriate vehicle
Fold induction = M free fatty acids SAMPLE
M free fatty acids VEHICLE
The R2 value should be equal or greater then 0.98 for the standard curve to be valid. Any R2 values below
0.98, must have the standard curve run again.

FREQUENTLY ASKED QUESTIONS
1. I do not have time to run the assay. Can I freeze the conditioned media in PLATE B? How long canI store the samples before I complete the assay? Yes. The conditioned media in PLATE B can be
immediately stored at -80 C for a maximum of 7 days. Bring the conditioned media in PLATE B to room
temperature BEFORE adding the FFA Reagents A and B and completing the assay.

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APPENDIX A: PLATE LAYOUT
ABCDEFGH

123456789101112Rev 8/15/2008

Page 7 of 8APPENDIX B: LIP-2 PROCEDURE FLOWCHART
Remove 150 l of the shipping medium andplace in your incubator for 5-7 days
(3-5 days for international customers)

ON DAY OF ASSAY
Make all test compound dilutions inAssay Buffer.

Remove 120 l media from all wells.
Add 200 l Wash Buffer to all wells.

Plate A

120 l media

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

200 l Wash Buffer
Plate A

Remove 120 l media & Wash
Buffer. Add another 200 l Wash
Buffer to all wells.

200 l Wash BufferOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Add another 200 lWash BufferPlate A

Remove all media & Wash Buffer. Add 75 ltreatments/controls to 3 wells at a time.

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Remove 3 wells at a time
Add treatments 3 wells at a timeoIncubate 3-5 hours at 37 C.
Plate A

Remove 50 l/well conditioned media from Plate A
to one of the blank assay plates providedReconstitute FFA Reagent A using Diluent A.
Add 100 l/well. Incubate 10 minutes @ 37 C.

Reconstitute FFA Reagent B using Diluent B.
Add 50 l/well. Incubate 10 minutes @ 37 C.

Place at room temp. for 5 minutes. Pop any
bubbles in each well using a clean pipet tip or large
gauge needle.

Measure the optical density of each wellat 540 nm using a spectrophotometer
plate reader.

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Page 8 of 8

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

blank plate
50 l

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

100 l/well
FFA Reagent A

50 l/well
FFA Reagent B

OOO
OOO
OOO
OOO
OOO

OOO
OOO
OOO
OOO
OOO

An additional
plate may benecessary forthe assay ofstandards.