Adipocyte Lipolysis Assay Kit for
3T3-L1 Cells
CAT# LIP-1-L1; LIP-1-NCL1
INSTRUCTION MANUALZBM0008.01

STORAGE CONDITIONS
96-well plate cultured 3T3-L1 preadipocytes (LIP-1-L1) 37°C incubator
Glycerol Reagent A & Buffers:

4°C

Glycerol Standard & Controls: -20°C
Long-term storage: If you ordered a kit with REAGENTS ONLY (cat# LIP-1-NCL1), 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 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 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)(919) 547-0693

Toll Free

1-866-ADIPOSE

Electronic mail (e-mail)World Wide Web

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

Rev. 8/18/2008

Page 1 of 11(866)-234-7673

INTRODUCTION
Lipolysis plays a central role in the regulation of energy balance. Lipolysis is the process inwhich triglycerides (TG) are hydrolyzed into glycerol and free fatty acids. This process releases freefatty 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. Elevated adipocyte lipolysis has been
observed in obese and diabetic individuals (Arner 1996). Excessive free fatty acid production isbelieved 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 activation of adenylate cyclase (AC), whichthen 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, thencatalyzes the hydrolysis 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 adenosine
monophosphate). 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 in cultured adipocytes.
EPINEPHRINE

1,NOREPINEPHRINE

2,

3 AR

AC

Gs

IR
PDE

ATP

ABBREVIATIONS:
AC
adenylate cyclase
AMP adenosine monophosphateARadrenergic receptors
ATPadenosine triphosphateGs
G protein coupled receptor
IR
insulin receptor
FFA
free fatty acids
PDE
phosphodiesterase
PKA
protein kinasePer
perilipins
TG
triglyceride
AMP adenosine monophosphateATPadenosine triphosphateIRinsulin receptor
PDEphosphodiesterasePer
perilipins

cAMP

P

5’-AMP
PKA

TG

Per

HSL

FFA + glycerol

FFA + glycerol

bloodstream

Figure 1. Overview of adipocyte lipolysis
Rev. 8/18/2008

Page 2 of 11

PRINCIPLE OF THE ASSAY
Lipolytic activity is assessed by the measurement of glycerol released into the medium from triglyceride
breakdown. Glycerol released to the medium is phosphorylated by adenosine triphosphate (ATP) forming
glycerol-1-phosphate (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) and hydrogen
peroxide (H2O2). A quinoneimine dye is produced by the peroxidase catalyzed coupling of 4-aminoantipyrine (4AAP) and sodium N-ethytl-N-(3-sulfopropyl)m-anisidine (ESPA) with H2O2, which shows an absorbancemaximum 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

ITEMS INCLUDED IN THE KIT
Cap
Color
---

UNIT

QTY

STORAGE

PLATE

1

37°C

---

PLATE

2

-----

BOTTLE

14°C

3T3-L1 Adipocyte Differentiation Medium (cat#DM-2-L1); 15ml (LIP-1-L1 ONLY)

BOTTLE

14°C

BOTTLE

1

4°C

Assay Buffer

3T3-L1 Adipocyte Maintenance Medium (cat# AM1-L1); 100ml (LIP-1-L1 ONLY)
100 ml

---BOTTLE

1

4°C

Wash Buffer

50 ml

---

BOTTLE

14°C

Vehicle0.1% DMSO in Assay Buffer

GREEN

1 ml /

1

-20°C

1

-20°C

1

-20°C

BOTTLE

1

4°C

EACH

2

-----

50 l /

1

-20°C

ITEM
Plate A
Assay Plates, blank
Preadipocyte
MediumDifferentiation
MediumAdipocyte Medium

DESCRIPTION
96 well plate 3T3-L1 preadipocytes(LIP-1-L1 ONLY)
96-well assay plate, blank (for samples +standards)
3T3-L1 Preadipocyte Medium (cat# PM-1-L1);
50ml (LIP-1-L1 ONLY)

VIAL
Positive Control

Alternate Positive
Control
Glycerol Reagent A
Tray
Glycerol standard

Rev. 8/18/2008

Isoproterenol, 10 mM in DMSO. Dilute to 1 M in
Assay Buffer before use! (i.e.1 l in 10 ml Assay
Buffer)3-Isobutyl-1-methylxanthine (IBMX), 100 mM in
DMSO Dilute to 100 M in Assay Buffer before
use! (i.e. 1 l in 1 ml Assay Buffer)
Reconstitute with 11 ml deionized water prior touse.
For multi-channel pipetters, clear polyvinyl

BLUE

Glycerol @ 1mM [Reconstitute with 200 l Wash
Buffer to make the 200 M glycerol standard; seepage 6 for recommended dilution scheme]

ORANGE

Page 3 of 11

10 l /VIAL

RED

10 l /VIAL

VIAL

Other equipment/reagents required but not provided with the kit:
Multi-channel pipet , single channel pipet and pipet tips
Sterile trays for multi-channel pipetters during differentiation of cells
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 diluting glycerol standards

NOTE:
THIS KIT IS DESIGNED FOR THE ASSAY OF A 96 WELL PLATE (100 ASSAY POINTS). IF YOU WISH TO TEST
ANOTHER PLATE FORMAT, PLEASE CONTACT ZEN-BIO TO PURCHASE ADDITIONAL REAGENTS TO COMPLETE YOURSTUDY.

ASSAY PROCEDURE
A. DIFFERENTIATION PROCEDURE
1. Preadipocytes are plated sub-confluent in 3T3-L1 Preadipocyte Medium (cat# PM-1-L1) and shipped thenext day via overnight delivery.
2. Incubate cells until they are 100% confluent (in about 4-5 days). Cells will need to be fed every other daywith PM-1-L1 during this time. See Table 1 for feeding volumes.
3. Once the cells are confluent, incubate an additional 48 hours before initiating differentiation.
4. Two days after the cells have been confluent, remove the Preadipocyte Medium (cat# PM-1-L1) andreplace with an appropriate volume 3T3-L1 Differentiation Medium (cat# DM-2-L1; see table 1 below forrecommended volumes). Incubate for 3 days.
5. Remove the 3T3-L1 Differentiation Medium and replace with 3T3-L1 Adipocyte Maintenance Medium.
Incubate for 2-3 days.
6. Feed cells every 2-3 days using 3T3-L1 Adipocyte Maintenance Medium until ready for assay. 3T3-L1
adipocytes are suitable for most assays 7-14 days post differentiation (see Table 1 and 3T3-L1 Growthand Differentiation Feeding Schedule)
Table 1. Feeding Volumes
Format96 well plate
48 well plate
24 well plate
12 well plate
6 well plate
T-75 flask
T-25 flask

Rev. 8/18/2008

Change PM-1-L1 to
PM-1-L1
OUT
IN

Change PM-1-L1 to
DM-2-L1
OUT
IN

Change DM-2-L1 to
AM-1-L1
OUT
IN

Change AM-1-L1 to
AM-1-L1OUT
IN

90 l/well
300 l /well
0.6 ml/well
1.2 ml/well
1.8 ml/well
12 ml/flask
4.2 ml/flask

150 l/well
500 l /well
1.0 ml/well
2.0 ml/well
3.0 ml/well
20 ml/flask
7 ml/flask

90 l /well
300 l /well
0.6 ml/well
1.2 ml/well
1.8 ml/well
12 ml/flask
4.2 ml/flask

90 l /well
300 l /well
0.6 ml/well
1.2 ml/well
1.8 ml/well
12 ml/flask
4.2 ml/flask

90 l/well
300 l /well
0.6 ml/well
1.2 ml/well
1.8 ml/well
12 ml/flask
4.2 ml/flask

150 l / well
500 l /well
1.0 ml/well
2.0 ml/well
3.0 ml/well
20 ml/flask
7 ml/flask

Page 4 of 11

120 l /well
400 l /well
0.8 ml/well
1.6 ml/well
2.4 ml/well
16 ml/flask
5.6 ml/flask

120 l /well
400 l /well
0.8 ml/well
1.6 ml/well
2.4 ml/well
16 ml/flask
5.6 ml/flask

3T3-L1 Growth and Differentiation Feeding Schedule
DAY

DAY

DAY

DAY

DAY

DAY

DAY

DAY

DAY

-2

0

3

5

7**

9

11

13

15

proliferationFeed
PM-1-L1

Feed
PM-1-L1

Feed
PM-1-L1

48 hrs100%
confluent*Feed
DM-2-L1

Feed
AM-1-L1

Feed
AM-1-L1

Feed
AM-1-L1

PREADIPOCYTE

Feed
AM-1-L1

Feed
AM-1-L1

Feed
AM-1-L1

MATURE ADIPOCYTE

nucleus
Lipid droplets
nucleus* Once the cells are 100% confluent, incubate an additional 48 hours before initiating differentiation.
** 3T3-L1 adipocytes are suitable for most assays 7-14 days post differentiation

B. LIPOLYSIS PROCEDURE
1. Make your stock solution using whatever vehicle is appropriate for your test compounds. Dilute

your 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 positive
controls. Include the Assay Buffer alone as a vehicle control. PLEASE NOTE: ZEN-BIO DOESNOT RECOMMEND THE USE OF SOLVENTS AT CONCENTRATIONS ABOVE 1%.
2. 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.

3. Remove all the media and Wash Buffer from the cells in triplicate wells. Treat the cells with 150

l of the test compounds resuspended in Assay Buffer three (3) wells at a time. Treat with thediluted IBMX and Isoproterenol as positive controls. Use the Assay Buffer alone as one of thevehicle 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 intriplicate.
4. OPTION: to determine if the compound alone reacts with the Glycerol Reagent A, prepare a

fresh plate (not included in kit) containing 100 l of the compound. This plate can be incubatedat 37oC with the treated cells. When performing the assay, add 100 l of Glycerol Reagent A
following the instructions in Steps 9 and 10.
Rev. 8/18/2008

Page 5 of 11°

5. Incubate the plates at 37 C in a humidified incubator for 5 hours (for time course experiments

the longest time point is usually 24 hours).
6. 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

7. Also at this time prepare the Glycerol Reagent A by adding 11ml 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).
8. At the end of the incubation, 100

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 100 l of each glycerol standard to any remaining empty wells in Plate B or use Plate C for
the standards.

9. Add the reconstituted Glycerol Reagent A solution to one of the disposable trays provided in thekit. Add 100 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.
10. The optical density of each well is then measured at 540 nm.
Rev. 8/18/2008

Page 6 of 11

GLYCEROL STANDARD CURVE
Generate standard curve: see example below[DO NOT use this standard curve to generate your data. This is an example.]

Zero (blank)= .040M Glycerol
3.125
6.25
12.5
25
50
100
200

OD
0.054
0.066
0.082
0.138
0.214
0.402
0.711

slope =
intercept=
r 2=

0.00340.00750.9985OD - blank
0.014
0.026
0.042
0.098
0.174
0.362
0.671

OD

Subtract the OD value of the 0 M standard from all OD values including the standard curve.

Standard Curve

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
Glycerol in uM

150

200

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 the proper signfor 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 reassayed using a lower dose of the compound at treatment OR a dilute solution of the conditioned medium at thetime of the assay.
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.
Data are expressed as M glycerol released.
OPTION: express data as Fold induction over appropriate vehicle
Fold induction = M glycerol SAMPLE
M glycerol VEHICLE

Rev. 8/18/2008

Page 7 of 11

TROUBLESHOOTING
ProblemHigh background or the glycerol reagent A
turns purple before the assay begins.

Suggestions
Use clean tray and tips
Change pipet tips frequently
Use Glycerol Reagent A before the expiration date

No response to either positive control

Visually observe adequate differentiation of thecultured adipocytes prior to assay.

Edge effects

Ensure a saturated humidity in the incubator toprevent evaporation from the outside wells

Inconsistent OD reading

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 the plate again.

FREQUENTLY ASKED QUESTIONS
1. 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 timecourse experiments, add 250

l assay medium with treatments per well. Remove 100

l foreach time point. We do not recommend modification of the 100 l required for the assay.
2. I have more samples plus standards to run than can fit on 1 96 well plate. Can I compare

data obtained from multiple plates? The lipolysis kit is designed for the assay of a single
plate. You may purchase 2 or more kits of the same lot number. You may then use one platethat includes the blank, vehicle(s), and positive and negative controls. Additional plates may
then be used for the assay of the remainder of your samples. In order to obtain comparable
data, all plates must be assayed on the same day using kits from the same lot number. Plate C
is provided for the assay of glycerol standards.
3. I do not have time to perform the assay. Can I freeze the conditioned media in PLATE B?

How long can I 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 Lipolysis Reagent and completingthe assay.

Rev. 8/18/2008

Page 8 of 11

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. 8/18/2008

Page 9 of 11

APPENDIX B: PROCEDURE FLOWCHART
Make
test compound
dilutions in
ONallDAYOF ASSAY
Assay Buffer.

Plate A = plate of mature 3T3-L1 adipocytes
Plate A

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

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.

200 l Wash Buffer

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Add another 200 l
Wash Buffer

Plate A

Remove all media & Wash Buffer. Add 150 ltreatments/controls to 3 wells at a time. OPTION: Add
compounds to a fresh plate without cells.

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

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

Incubate 5-24 hours at 37oC.

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

Remove 100 l/well conditioned media
from Plate A to Plate B. Add 100 lglycerol standards to empty wells

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Plate B
100 l

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Blank assay plate

Add 100 l/well reconstituted Glycerol Reagent A
to Plate B (including the glycerol standards at
100 l/well in the blank assay plate(s) and optional
plate without cells.
Incubate at 25oC (room temperature)for 15 minutes. Pop the bubbles ineach well.

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

Rev. 8/18/2008

Page 10 of 11

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

Blank assay plate
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.

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.
10. Tansey JT, Sztalryd C, Hlavin EM, Kimmel AR, Londos C. (2004) IUBMB Life 56(7): 379-85.

Rev. 8/18/2008

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