Adipose Tissue Explant
Lipolysis Assay Kit: Glycerol Detection
Cat# LIP-6-NC

INSTRUCTION MANUALZBM0046.03

STORAGE CONDITIONSAll orders are delivered via Federal Express Priority courier at room temperature.
All orders must be processed immediately upon arrival.
Glycerol Reagent A & Buffers:
Store at 4C.
Use reconstituted Glycerol Reagent A within 7 days.
Glycerol Standards & Controls:
Store at -20°C
All Zen-Bio Inc products are for research use only. Not approved for human or veterinary use orfor use in diagnostic or clinical procedures.
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)(919) 547-0693
Toll Free
1-866-ADIPOSE



Electronic mail (e-mail)World Wide Web

Rev Nov.2010(866)-234-7673

information@zen-bio.com
http://www.zenbio.com
Page 1 of 11

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 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 whole
mammalian adipose tissue pieces.

Figure 1. Overview of adipocyte lipolysis

EPINEPHRINE1 , 2 ,3 AR

NOREPINEPHRINEACGsATPIRPDEcAMP

P

5’-AMPPKATG

PerHSL

FFA + glycerol

FFA + glycerol

bloodstream
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Page 2 of 11

ABBREVIATIONS:ACadenylate cyclaseARadrenergic receptors
G protein coupled receptor
Gs
FFA
free fatty acids
PKA
protein kinase
AMP adenosine monophosphateATPadenosine 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 + O2 G-1-P + ADP

DAP + H2O2

H2O2 + 4-AAP + ESPA Quinoneimine dye + H2O

ITEMS INCLUDED IN THE KITITEM

DESCRIPTIONBlank Assay Plate
Assay Buffer
Wash BufferVehicle

96-well assay plate, blank
100 ml100 ml
0.1% DMSO in Assay Buffer

Positive control

Glycerol Reagent A
Tray
Glycerol standard
ALTERNATE :Positive control
4CapColor-------QTY

STORAGE

PLATE

1112

----4°C
4°C-20°C

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 11.0 ml deionized water prior touse. Use within 7 days!
Clear polyvinyl, 50ml capacity

BLUE10 l /VIAL

1-20°C

BOTTLE

14°C

EACHGlycerol @ 1mM [Reconstitute with 400 l Wash
Buffer to make the 200 M glycerol standard; seepage 5 for recommended dilution scheme]
3-Isobutyl-1-methylxanthine (IBMX), 100 mM inDMSO Dilute to 100 M in Assay Buffer beforeuse! (i.e.5 l in 5 ml Assay Buffer).

ORANGE100 l/ VIAL

2
1-----20°CRED

10 l /VIAL

1-20°C

Other equipment/reagents required but not provided with the kit: Flat bottom, Tissue culture treated cultureware, 24- or 48 well format
 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, blank Tubes for dilution of standards

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UNITPage 3 of 11

ASSAY 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 positivecontrols. Include the Assay Buffer alone as a vehicle control. PLEASE NOTE: ZEN-BIO DOESNOT RECOMMEND THE USE OF SOLVENTS AT CONCENTRATIONS ABOVE 1%.
2. Wash fresh non-enzymatically digested adipose tissue using Wash Buffer. If isolating multiplesamples, store removed adipose tissue at room temperature in sterile containers (not provided)containing sufficient phosphate buffered saline (PBS) or Wash Buffer to completely submerge thesample until ready for assay.
3. Place a 24 or 48 well plate on a digital balance and tare the unit. While holding the adipose tissue

chunk using tissue forceps, use scissors to cut small uniform pieces (by weight) of adipose tissue
directly into each well of the plate. Record weight per well. Suggested weights are 100mg per wellfor a 24 well plate or 50mg for a 48 well plate.
4. Mince tissue for 1 minute using sharp scissors.
5. Treat each well with 300-500 l (48 well-24 well) of the test compounds resuspended in Assay

Buffer three (3) wells at a time. Treat with the diluted Isoproterenol or optionally, IBMX, as
positive control. Use the Assay Buffer alone as one of the vehicle controls. Please be sure toinclude both the vehicle provided in the kit and your vehicle (if your test compounds are notdissolved in DMSO).
6. OPTION: to determine if the compound alone reacts with the Glycerol Reagent A, prepare a freshplate (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 l of Glycerol Reagent A following theinstructions in Steps 10 and 11.
o

7. Incubate the plates at 37 C-humidified incubator for 5-24 hours (for time course experiments thelongest time point is usually 24 hours).

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8. One hour prior to the assay, prepare the glycerol standards as follows:

Briefly spin down the contents of the glycerol standard tube before reconstitution. Pipette400 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 250 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.

400 lWash
Buffer250 l250 l 250 l 250 l

250 l 250 l

Std200
M100
M50
M25
M

12.5
M

6.25
M

3.125
M

Note: The above dilution series generates enough volume to perform the standard curve induplicate. If you wish to perform the standard curve in duplicate, please note that eight fewer data
points can be assayed with this kit.
9. 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).
10. At the end of the incubation, a small sample of the conditioned media is removed and transferredto the provided 96 well plate. The sample may need to be diluted using Assay Buffer. We
suggest a 2-5 fold dilution with a total volume of 100l per well (i.e. 20l conditioned medium +800l Assay Buffer= dilution factor of 5). Add 100 l of each glycerol standard to any remaining
empty wells the blank 96-well assay plate.
11. 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 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.
12. 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.
uM
glycerol

ODblank

ODblank

Avg
ODblank

ODOD

0

0.044

0.041

3.125

0.054

0.053

0.012

0.011

0.011

6.25

0.062

0.063

0.020

0.021

0.020

12.5

0.083

0.084

0.041

0.042

0.041

25

0.126

0.125

0.084

0.083

0.083

50

0.205

0.208

0.163

0.166

0.164

100

0.372

0.374

0.330

0.332

0.331

200

0.698

0.697

0.656

0.655

0.655

Slope

0.003

Intercept

0.001

2

R

0.043

1.000

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 – (0.001))/0.003 where 0.003= slope of the line and 0.001= 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. Multiply values by the dilution factor (if used).
OPTION: express data as Fold induction over appropriate vehicle
Fold induction =M glycerol SAMPLEM glycerol VEHICLE

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TROUBLESHOOTING
ProblemHigh 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 Use a new pipet tips for each well during transfer ofconditioned medium.
 Do keep samples at room temperature until ready for assay

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.
 Make sure adipose tissue sizes are as uniform as possible.

FREQUENTLY ASKED QUESTIONS
1. When do I need to use the IBMX positive control? If you use the 3-5 hour incubation described inthis manual, you will not need to use the IBMX as your positive control. The IBMX positive control isdesigned for treatments ranging from 5-24 hours. The IBMX alternate control may be used in addition
to the Isoproterenol 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 500-750 l assay medium with treatments per well. Remove 50 l for each time point. Complete
the assay using 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. Youmay 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 remainderof your samples assayed. In order to obtain comparable data, both plates must be assayed on thesame day using kits and cells 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

one of the assay plates provided with the kit? How long can I store the samples before I
complete the assay? Yes. The conditioned media can be immediately stored at -80C for amaximum of 7 days. Bring the conditioned media in the plate to room temperature BEFORE adding
the Glycerol Reagent A and completing the assay.

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APPENDIX A: PLATE MAPS
24 WELL
1A 1223344556B 789101112C 131415161718

D 1920212223

24

48-WELL
1212A6345678345678B 910111213

141516C 17

181920

21

22

23

24

D 25

262728

2930

31

32

E 33

34

35

36

37

38

39

40

F 41

42

43

44

45

46

47

48

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96-WELL.

ABCDEFGH

123456789101112Rev Nov.2010

Page 9 of 11

APPENDIX B: PROCEDURE FLOWCHART
Isolate fresh adipose tissue. Store atroom temperature in Wash Buffer orPBS.

Make all test compound dilutions inAssay Buffer.

Add 300-500 l treatments/controls to 3 wells at a time.

Plate AooooooooooooooooooooooooOPTION: Add 50 l/well compounds to a fresh plate
without cells.

Incubate 5-24 hours at 37oC.

One hour prior to assay, reconstitute
Glycerol Reagent A and preparestandards. Keep all at room temp.
Remove conditioned media from Plate
A to a blank assay plate.
Dilute with Assay Buffer up to 100 µl.
Add 100 l/well glycerol standards toempty wells.

Add 100 l/well reconstituted Glycerol Reagent Ato the plate (including the glycerol standards at100l/well ) and optional plate without cells.

Incubate at 25oC (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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Plate Aoooooooooooooooooo

OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO

blank plate
5-50 lOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOGLYCEROLREAGENT A

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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Rev Nov.2010

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