Cultured Human Adipocyte Lipolysis Assay Kit
Cat# LIP-1; LIP-1-NC
INSTRUCTION MANUALZBM0002.01

STORAGE CONDITIONSAll 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

Assay Plate A: Cultured human adipocytes (LIP-1 kit ONLY)
37°C CO2 incubator
Glycerol Reagent A & Buffers:
Store at 4 C.
Glycerol Standards & Controls:
Store at -20°C
Long-term storage:
LIP-1-NC (Reagents Only) kit: remove the glycerol reagent A from the box and place at 4°C, store
the rest of the kit at -20°C. Reagents are good for 6 months if stored properly.
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 ofthe use, 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 10(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 othertissues and exert other effects throughout the body. Elevated adipocyte lipolysis has been observed inobese and diabetic individuals (Arner 1996). Excessive free fatty acid production is believed tocontribute 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 bymediating 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 theintracellular Gs proteins in adipocytes. This leads to the activation of adenylate cyclase (AC), which thenincreases 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 thehydrolysis of triglycerides and results in the release of glycerol and FFA (increased lipolysis).
Phosphodiesterases (PDE) are enzymes that hydrolyze cAMP to 5’-AMP (5 prime adenosinemonophosphate). This action results in a decrease in lipolysis. PDE inhibitors increase intracellularcAMP 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
affect lipolysis via -adrenergic receptors (Robidoux et al. 2004).
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

AC

Gs

IR
PDE

ATPcAMPP

5’-AMP
PKATG

Per

HSL

FFA + glycerol

FFA + glycerol

bloodstream
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ABBREVIATIONS:ACadenylate cyclaseARadrenergic receptors
Gs
G protein coupled receptor
FFA
free fatty acids
PKA
protein kinase
AMP adenosine monophosphate
ATPadenosine triphosphate
IRinsulin receptor
PDEphosphodiesteraseTG
triglyceridePRINCIPLE OF THE ASSAY
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 isthen oxidized by glycerol phosphate oxidase to dihydroxyacetone phosphate (DAP) and hydrogen peroxide(H2O2). A quinoeimine dye is produced by the peroxidase catalyzed coupling of 4-aminoantipyrine (4-AAP) andsodium N-ethyl-N-(3-sulfopropyl)m-anisidine (ESPA) with H2O2, which shows 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 + ADP

G-1-P + O2

DAP + H2O2

H2O2 + 4-AAP + ESPA

Quinoneimine dye + H2O

ITEMS INCLUDED IN THE KITITEM

DESCRIPTION

Adipocytes, Plate A
Blank Assay Plates
Assay Buffer
Wash Buffer
VehicleCultured human subcutaneous adipocytes
96-well assay plates, blank
100 ml50 ml
0.1% DMSO in Assay Buffer

Positive control

Glycerol Reagent ATrayGlycerol standard

ALTERNATE :Positive control

UNIT

QTY

STORAGE

PLATE1211137°C----4°C
4°C
-20°C

PLATE
BOTTLEBOTTLEGREEN

1 ml /
VIAL

Isoproterenol, 10 mM in DMSO. Dilute to 1 M in
Assay Buffer before use! (i.e.1 l in 10 ml AssayBuffer)
Reconstitute with 11 ml deionized water prior touse.
For multi-channel pipetters, clear polyvinylBLUE

10 l /
VIAL

1-20°C

BOTTLE

14°C

EACH

Glycerol @ 1mM [Reconstitute with 200 l Wash
Buffer to make the 200 M glycerol standard; seepage 6 for recommended dilution scheme]
3-Isobutyl-1-methylxanthine (IBMX), 100 mM inDMSO Dilute to 100 M in Assay Buffer before
use! (i.e. 1 l in 1 ml Assay Buffer).
USE ONLY IF YOUR TREATMENT TIME
EXCEEDS 5 HOURS.

ORANGE50 l /VIAL

2
1-----20°C

RED

10 l /VIAL

1-20°C

4

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
Option – Step 5 of Assay Procedure: 96 well plate, blankTubes for dilution of standards
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CapColor---------Page 3 of 10

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. Diluteyour stock solutions to their final concentration in Assay Buffer (100 ml is available). NOTE: if
desired, maintain a constant concentration of solvent by preparing all compound dilutions in thehighest concentration of that solvent. Dilute your controls in assay buffer. Prepare all vehicles asappropriate for your compounds, 0.1% DMSO has been included as the vehicle for the positivecontrols. Include the Assay Buffer alone as a vehicle control. PLEASE NOTE: ZEN-BIO DOESNOT 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 200l 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 or optionally, IBMX (for treatments 5-24 hours), as positive control. Use theAssay Buffer alone as one of the vehicle controls. Please be sure to include both the vehicle
provided in the kit and your vehicle (if your test compounds are not dissolved in DMSO). The
assay should be performed in triplicate.
5. 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 37 oC
with the treated cells. When performing the assay, add 50 l of Glycerol Reagent A following theinstructions in Steps 10 and 11.
6. Incubate the plates at 37oC-humidified incubator for 3 hours (for time course experiments thelongest time point is usually 24 hours).
Rev 8/15/2008

Page 4 of 107. 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, and thewash buffer serves as the zero standard.

200 l
Wash
Buffer

125 l

125 l 125 l 125 l 125 l

125 l

Std200
M

100
M

50
M

25
M

12.5
M

6.25
M

3.125
M

8. Also at this time prepare the Glycerol Reagent A by adding 11 ml room temperature deionizedwater per bottle and gently invert. DO NOT VORTEX! Use a pipet to ensure that the powder iscompletely dissolved. 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 protectedbottle. Reconstituted Glycerol Reagent A is stable for 60 days refrigerated (2-8 C); store any
remaining solution refrigerated (2-8 C).
9. At the end of the incubation, 50

l of the conditioned media is removed and transferred to thecorresponding well of Plate B. [This is most easily accomplished using a multi-channel pipet.]
Add 50l of each glycerol standard to any remaining empty wells in one of the blank assay

plates.
10. 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 the assay plates containing samples. Gently, pipet up
and down once to mix. Pop the bubbles using a large gauge needle or a clean pipet tip. The
plate is then incubated at 25oC (room temperature) for 15 minutes.
11. The optical density of each well is then measured at 540 nm.
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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

Standard Curve= .040
OD
0.054
0.066
0.082
0.138
0.214
0.402
0.711

OD blank
0.014
0.026
0.042
0.098
0.174
0.3620.671

y = 0.0034x + 0.0075
R2 = 0.9985

0.80.6OD

Zero(blank)

0.4
0.2
0.0050

100

150

200

250

Glycerol in uM

slope =0.0034
intercept= 0.0075
R2=0.9985

y = 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 the following
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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TROUBLESHOOTINGProblem
High background or the glycerol
reagent A turns purple before theassay begins.
No response to positive control

Suggestions
Change pipet tips frequently
Use Glycerol Reagent A before the expiration date
Do not add the compounds and controls too fast. The cells
can float if a solution is added too fast.
Make sure to starve the cells for 5-7 days BEFORE initiatingtreatment.
DO NOT use IBMX as the positive control if you areincubating for less than 5 hours.

Edge effects
Inconsistent OD reading

Ensure a saturated humidity in the incubator to prevent
evaporation from the outside wells
The Assay Buffer contains bovine serum albumin (BSA). Be
careful when pipetting to avoid bubbles. If bubbles persist,burst the bubbles using a large gauge needle and read theplate again.

FREQUENTLY ASKED QUESTIONS
1. When do I need to use the IBMX positive control? If you use the 3-5 hour incubation described in thismanual, you will not need to use the IBMX as your positive control. The IBMX positive control is designed
for treatments ranging from 5-24 hours. The IBMX alternate control may be used in addition to theIsoproterenol positive control if your treatment time will exceed 5 hours.
2. I want to perform a lipolysis time course experiment. How many time points can I complete ?

We do not recommend performing more than 2 time points per assay. For time course experiments, add
125 l assay medium with treatments per well. Remove 50 l for each time point. Complete the assayusing an equal volume Glycerol Reagent A.
3. I have more samples plus standards to run than can fit on 1 96 well plate. Can I compare dataobtained from multiple plates? The lipolysis kit is designed for the assay of a single plate. You may
purchase 2 kits of the same lot number. You may then use one plate that includes the blank, vehicle(s),and positive and negative controls. The second plate may then be used for the remainder of your samples
assayed. In order to obtain comparable data, both plates must be assayed on the same day using kits andcells from the same lot number. An additional blank assay plate is provided for the assay of glycerol
standards.
4. I do not have time to pop the bubbles and read the plate. Can I freeze the conditioned media in oneof the assay plates provided with the kit? How long can I store the samples before I complete theassay? Yes. The conditioned media can be immediately stored at -80 C for a maximum of 7 days. Bringthe conditioned media in the plate to room temperature BEFORE adding the Glycerol Reagent A andcompleting the assay.
Rev 8/15/2008

Page 7 of 10APPENDIX A: PLATE LAYOUT.

ABCDEFGH

123456789101112

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Page 8 of 10

APPENDIX B: PROCEDURE FLOWCHART
Remove 150 l of the shipping medium
and place in your incubator for 5-7 days
(3-5 days for international cutomers)

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

Remove all media & Wash Buffer. Add 75 ltreatments/controls to 3 wells at a time. OPTION: Add 50l/well compounds to a fresh plate without cells.

200 l Wash Buffer

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Add another 200 lWash Buffer

Plate AOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Remove 3 wells at a time
Add treatments 3 wells at a time

oIncubate 3-5 hours at 37 C.

One hour prior to assay, reconstitute
Glycerol Reagent A and preparestandards. Keep all at room temp.
Plate A

Remove 50 l/well conditioned media
from Plate A to a blank assay plate.
Add 50 l/well glycerol standards toempty wells.

Add 50 l/well reconstituted Glycerol Reagent A
to the plate (including the glycerol standards at50 l/well ) and optional plate without cells.

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

Measure the optical density of eachwell at 540 nm using aspectrophotometer plate reader.

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OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

blank plate
50 l

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOGLYCEROLREAGENT A

OOO
OOO
OOO
OOO
OOO

An additional blank assay plate
may be necessary for the assay ofglycerol standards if al 96 wells
are used.

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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