Cultured Human Adipocyte Lipolysis Assay
Kit for Detection of Both Free Glycerol and
Non-Esterified Fatty Acids
CAT# LIP-3; LIP-3-NC
INSTRUCTION MANUALZBM0011.03

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 ZenBio, Inc. Zen-Bio, Inc. shall have no liability for any direct, indirect, consequential, or incidental damages arising out of theuse, the results of use, or the inability to use this product.

ORDERING INFORMATION AND TECHNICAL SERVICES
Zen-Bio, Inc.
3200 Chapel Hill-Nelson Blvd., Suite 104
PO Box 13888
Research Triangle Park, NC 27709
Telephone(919) 547-0692

Facsimile (FAX)
Toll Free(919) 547-0693
1-866-ADIPOSE

Electronic mail (e-mail)World Wide Web

information@zen-bio.com
http://www.zenbio.com

Rev 8/15/2008

Page 1 of 12(866)-234-7673

INTRODUCTION
Lipolysis plays a central role in the regulation of energy balance. Lipolysis is the process
in which triglycerides (TG) are hydrolyzed into glycerol and free fatty acids. This process
releases free fatty acids (FFA) into the bloodstream where they may be either re-esterified by
the adipocyte or travel to other tissues and exert other effects throughout the body. Elevatedadipocyte lipolysis has been observed in obese and diabetic individuals (Arner 1996).
Excessive free fatty acid production is believed to contribute to insulin resistance in skeletal
muscle that is observed in obesity. Hormone sensitive lipase is the rate-limiting enzyme
catalyzing triglyceride breakdown. Perilipins, one of the PAT (perilipins, adipophilin, TIP47
proteins) family of lipid-associated proteins, are implicated in adipocyte lipolysis by mediating
the interaction of HSL with the triacylglycerol molecule (Brasaemle et al. 2004; reviewed in,
Tansey et al. 2004.) The presence of these proteins corresponds to lipolytic stimulation in
cultured adipocytes (Braemle et al. 2004).
The sympathetic nervous system also 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 proteins in adipocytes. This leads to the activationof 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 rate-limiting
enzyme regulating adipocyte lipolysis, then catalyzes the hydrolysis of triglycerides and resultsin the release of glycerol and FFA (increased lipolysis). Phosphodiesterases (PDE) are
enzymes that hydrolyze cAMP to 5’-AMP (5 prime adenosine monophosphate). This action
results in a decrease in lipolysis. PDE inhibitors increase intracellular cAMP levels. 3-isobutyl1-methylxanthine (IBMX), a non-specific inhibitor of cAMP phosphodiesterases (PDE), is usedas the positive control if your test compounds are suspected PDE inhibitors. Isoproterenol, anon-specific -adrenergic agonist is used as the positive control if your test compounds affectlipolysis via -adrenergic receptors (Robidoux et al. 2004).
This lipolysis assay kit provides the tool to study chemical compounds that may
influence lipolysis in cultured human adipocytes.
Figure 1. Overview of adipocyte lipolysis
EPINEPHRINE

1,NOREPINEPHRINE

2,

3 AR

AC

Gs
PDE

ATP

ABBREVIATIONS:
AC
adenylate cyclaseARadrenergic receptors
Gs
G protein coupled receptor
FFA
free fatty acids
PKA
protein kinase
AMP adenosine monophosphateATPadenosine triphosphateIRinsulin receptor
PDEphosphodiesteraseTG
triglyceride

IR

cAMP

P

5’-AMP
PKA

TG

Per

HSL

FFA + glycerol

FFA + glycerol

Rev 8/15/2008

bloodstream

Page 2 of 12

PRINCIPLES OF THE ASSAYS
Detection of Free Glycerol
Assessing lipolytic activity by the measurement of glycerol released into the medium. Glycerol
released to the medium is phosphorylated by adenosine triphosphate (ATP) forming glycerol-1phosphate (G-1-P) and adenosine-5’-diphosphate (ADP) in the reaction catalyzed by glycerol kinase.
G-1-P is then oxidized by glycerol phosphate oxidase to dihydroxyacetone phosphate (DAP) andhydrogen peroxide (H2O2). A quinoneimine dye is produced by the peroxidase catalyzed coupling of 4aminoantipyrine (4-AAP) and sodium N-ethytl-N-(3-sulfopropyl)m-anisidine (ESPA) with H2O2, whichshows an absorbance maximum at 540nm.

The increase in absorbance at 540nm is directly

proportional to glycerol concentration of the sample.
GLYCEROL + ATP
G-1-P + O2

G-1-P + ADP
DAP + H2O2

H2O2 +4-AAP + ESPA

Quinoneimine dye + H2O

Detection of Non-Esterified Fatty Acids (Free Fatty Acids; FFA)
Assessment of lipolytic activity can also be detected through a coupled reaction to measure nonEsterified 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 thereaction. The acyl-CoA derivatives react withACS
HCOOH + ATP + CoA
Acyl-CoA + AMP + PPi
oxygen in the presence of acyl-CoA oxidase(NEFA)(ACOD) to produce hydrogen peroxide.
Hydrogen peroxide in the presence of
ACOD
Acyl-CoA + O2
2,3-trans-Enoyl-CoA + H2O2
peroxidase (POD) allows the oxidative
condensationof
3-methyl-N-ethyl-N-( CHNHhydroxyethyl)-aniline with 4-aminoantipyrineNN
C H OH
which forms a purple product that absorbs
N
O
CH
POD
N
N
+
O
N
N
light at 550nm. This allows the concentration 2H O ++ 4H OC H OHof NEFA to be determined from the opticaldensity measured at 540 - 550nm.
2

2

2

5

2

4

2

5

2

4

2

2

NOTE:
3 fatty acid molecules are released per triglyceride molecule resulting in a 3:1 fatty acidto glycerol concentration.

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ITEMS INCLUDED IN THE KITITEM

DESCRIPTION

Cap
Color

UNIT

QTY

STORAGE

Adipocytes, Plate A

Cultured human subcutaneous
adipocytes

---

PLATE

1

37°C

Assay Plates

96-well assay plate, blank

---

PLATE

3

-----

---

50ML

1

4°C

Wash Buffer
Vehicle0.1% DMSO in LIP-2 Assay Buffer

PURPLE

1 ml /
VIAL

1

-20°C

Positive control

Isoproterenol, 10 mM in DMSO. DiluteBLUE

10 l /

1-20°C

BOTTLE

1

4°C

EACH

4

-----

50 l /

1-20°C

to 1 M in Assay Buffer before use!

VIAL

(i.e.1 l in 10 ml Assay Buffer)
Glycerol Reagent A

Reconstitute with 11.0 ml deionizedwater prior to use.

Tray

For multi-channel pipetters, clear
polyvinyl

Glycerol standard

Glycerol @ 1mM [Dilute with 200 l---

ORANGE

VIAL

Wash Buffer to make the 200 M
glycerol standard; see page 6 forrecommended dilution scheme]
LIP2/3 Assay Buffer

100ml

FFA Standard

1mM Stock. See page 5 for standard
curve preparation

---

100ML

1

4°C

AMBER

100 l /

1

4°C

VIAL

FFA Diluent A

YELLOW

10.5ML

1

4°C

FFA Diluent B

PINK

5.5ML

1

4°C

YELLOW

BOTTLE

1

4°C

PINK

BOTTLE

14°C

FFA Reagent A
FFA Reagent B

Reconstitute using 10.5 ml FFA Diluent
A. Discard remainder after 10 days
Reconstitute using 5.5 ml FFA Diluent
B. Discard remainder after 10 days

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
96 well plate of adipocytes (LIP-3-NC) (cat# SA-1096)
Tubes for dilution of standards

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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
for the positive controls. Include the Assay Buffer alone as a vehicle control. PLEASE NOTE:ZEN-BIO DOES NOT RECOMMEND THE USE OF SOLVENTS AT CONCENTRATIONSABOVE 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 with100 l of the test compounds resuspended in Assay Buffer three (3) wells at a time. Treat withthe diluted 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 plate 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.

Rev 8/15/2008

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A. DETECTION OF NON-ESTERIFIED FATTY ACIDS
1. Prepare the standard curve using the FFA 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:
The kit standard solution is the 1.0 mM standard. Pipette 60 l of Dilution Buffer into 6
tubes (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 l

30 l 30 l 30 l 30 l

30 l

Std
FFA
Std

333
M

111
M

37
M

12.3
M

4.1
M

1.4
M

2. 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).
3. At the end of the incubation, 30 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 30 l of each standard to empty wells.
4. 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.
5. 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).
6. Add the reconstituted FFA Reagent B to the other disposable tray provided in the kit. Add 50 lof FFA Reagent B to each well. Gently shake the plate to ensure mixing. Place in a 37 oC
incubator for 10 minutes.
7. 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.
8. The optical density of each well is then measured at 540 nm.
Rev 8/15/2008

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B. DETECTION OF FREE GLYCEROL
1. One hour prior to the assay, prepare the glycerol standards as follows:
Briefly spin down the contents of the glycerol standard tube before reconstitution. Pipette
200 l of Wash Buffer into the 1 mM glycerol standard tube provided and mix well byvortexing. This produces a diluted stock glycerol standard of 200 M. Pipette 125 l ofwash buffer into 6 tubes (not provided). Using the newly diluted stock glycerol solution,
prepare a dilution series as depicted below. Mix each new dilution thoroughly before
proceeding to the next. The 200 M stock dilution serves as the highest standard, andthe wash buffer serves as the zero standard.

200 l
Wash
Buffer

125 l

125 l 125 l 125 l 125 l

125 l

Std
200
M

100
M

50
M

25
M

12.5
M

6.25
M

3.125
M

2. Also at this time prepare the Glycerol Reagent A by adding 11.0 ml room temperature deionizedwater per bottle and gently invert. DO NOT VORTEX! Use a pipet to ensure that the powder iscompletely dissolved. Store at room temperature. If using a Reagent A solution previously
prepared and stored at 2-8 C, also bring to room temperature. Make sure there is enough
Reagent A from one solution to treat all the points in the assay. It may be necessary to combine
solutions. Store in a light protected bottle. Reconstituted Glycerol Reagent A is stable for 60
days refrigerated (2-8 C); store any remaining solution refrigerated (2-8 C).
3. At the end of the incubation, an additional 50 l of the conditioned media is removed andtransferred to the corresponding well of a blank plate for assessment of free glycerol. [This ismost easily accomplished using a multi-channel pipet. Add 50 l of each glycerol standard to
any remaining empty wells in one of the blank assay plates.
4. OPTION: to determine if the compound alone reacts with the Glycerol Reagent A, prepare a
fresh plate (not included in kit) containing 50 l of the compound. This plate can be incubated at
37oC with the treated cells. When performing the assay, add 50
following the instructions in Steps 5 and 6.

l of Glycerol Reagent A

5. Add the reconstituted Glycerol Reagent A solution to one of the disposable trays provided in thekit. Add 50 l of Reagent A to each well of Plate B and Plate C (if used). Gently, pipet up anddown once to mix. Pop the bubbles using a large gauge needle or a clean pipet tip. The plate isthen incubated at 25oC (room temperature) for 15 minutes.
6. The optical density of each well is then measured at 540 nm.
Rev 8/15/2008

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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 4-parameter fit may be used to calculate standard curve.

Standard Curve
0.75

OD
OD - zero
0.68
0.636
0.244
0.2
0.104
0.06
0.063
0.019
0.05
0.006
0.046
0.002
0.044
0

0.50

O.D.

M
std
333
111
37
12.3
4.1
1.4
0

0.25

0.00
0

100

200

300

400

M standard

y = 0.0019x - 0.0045
R2 = 0.9995
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.

Rev 8/15/2008

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GLYCEROL 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.

M
Glycerol
3.125
6.25
12.5
25
50
100
200

= .040

OD
0.054
0.066
0.082
0.138
0.214
0.402
0.711

Standard Curve

OD blank
0.014
0.026
0.042
0.098
0.174
0.362
0.671

OD

Zero
(blank)

0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0

y = 0.0034x + 0.0075
R2 = 0.9985

0

50

100

150

200

250

Glycerol in uM

slope =
0.0034intercept= 0.0075
r2 =0.9985y = observed O.D. minus the blankx = concentration of glycerol in M
To calculate x for each y, (i.e. to change the observed O.D. into glycerol concentration) use thefollowing equation:y=(slope) times (x) plus intercept
y=mx+b so x=(y-b)/mx=(y – 0.0075)/0.003 where 0.003= slope of the line and 0.0075= y intercept. Be careful to enter theproper sign for the y intercept value as it may be a negative number.
Any OD values greater than the highest standard (200 M) should be suspect. The compound should
be re-assayed using a lower dose of the compound at treatment OR a dilute solution of the condition
medium at the time of the assay.
The R2 value should be equal or greater then 0.98 for the standard curve to be valid. Any R2values below 0.98, must have the standard curve run again.

Data are expressed as M glycerol released.
OPTION: express data as Fold induction over appropriate vehicle
Fold induction = M glycerol SAMPLE
M glycerol VEHICLE
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APPENDIX A: PLATE LAYOUT
.

A

B

C

D

E

F

G

H

1
2
3
4
5
6
7
8
9
10
11
12

Rev 8/15/2008

Page 10 of 12

APPENDIX B: 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 in
Assay Buffer.

Plate A

120 l media

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

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

200 l Wash Buffer
Plate A

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

200 l Wash Buffer

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Add another 200 l
Wash Buffer

Plate AOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Remove all media & Wash Buffer. Add 100 ltreatments/controls to 3 wells at a time.

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

FREE FATTY ACID DETECTION
Plate A

Remove 30 l/well conditioned media from
Plate A to Plate B.

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Reconstitute FFA Reagent A using Diluent A.
Add 100 l/well. Incubate 10 minutes @ 37 C.

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

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

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

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.

Rev 8/15/2008

Page 11 of 12

Assay Plate
30 l

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

100 l/well
FFA Reagent A

50 l/well
FFA Reagent B

OOO
OOO
OOO
OOO
OOO

OOO
OOO
OOO
OOO
OOO

Plate C may
be necessary
for the assay
of standards
if al 96 wellsof Plate A areused.

FREE GLYCEROL DETECTION
One hour prior to assay, reconstitute
Glycerol Reagent A and prepare standards.
Keep all at room temp.
Remove 50 l/well conditioned media
from Plate A to a blank assay plate. Add
50 l standards to empty wells.

Add 50 l/well reconstituted Glycerol Reagent A toa blank assay plate (including the glycerol
standards at 50 l/well and optional plate without
cells).

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

50 l

GLYCEROLREAGENT A

OOO
OOO
OOO
OOO
OOO

Plate C may be necessary for theassay of glycerol standards if al 96
wells of Plate A are used.

oIncubate at 25 C (room temperature)for 15 minutes. Pop the bubbles ineach well.

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

REFERENCES
1. Arner P (1996) Diabetes Rev 4(4):450-463.
2. Botion LM & Green A. Diabetes (1999) 48:1691-1697
3. Brasaemle DL, Dolios G, Shapiro L, Wang R. (2004) J Biol Chem 279(45): 46835-42.
4. Cooper DMF, Schlegel W, Lin MC, Rodbell M. (1979) J Biol Chem 254(18):8927-8931.
5. Dyck DJ Can J Appl Physiol (2000) 25(6):495-523.
6. Kordik CP & Reitz AB. J Medicinal Chem (1999) 42(2):181-201.
7. Rieusset J, Chambrier C, Bouzakri K, Dussere E, Auwerx J, Riou J-P, Laville M, Vidal H.
Diabetologia (2001) 44:544-554.
8. Robidoux J, Martin TL, Collins S. (2004) Ann Rev Chem 253: 7570-7578.
9. Scriba D, Aprath-Husmann I, Blum WF, Hauner H. Eur J Endocrinol (2000) 143:439-445
10. Snyder PB Emerging Therapeutic Targets (1999) 3(4): 587-599.
11. Tansey JT, Sztalryd C, Hlavin EM, Kimmel AR, Londos C. (2004) IUBMB Life 56(7):379-85.
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