ZBM0048.01 LIP3RB kit RV08.09 - Zen
Contents
1. Pipette 60 of Dilution Buffer Assay Buffer into 6 tubes not provided Pipette 30 of the FFA Standard Stock into a tube labeled 333 uM Prepare a dilution series as depicted below Mix each new dilution thoroughly before proceeding to the next The Assay Buffer alone serves as the zero standard Add 50ml 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 2 8 C At the end of the incubation 30 of the conditioned media is removed and transferred to the corresponding well of a blank plate for assessment of non esterified fatty acids This is most easily accomplished using a multi channel pipet Add 30 of each standard to empty wells Add the reconstituted FFA Reagent A to one of the disposable trays provided in the kit Add 100 of FFA Reagent A to each well Gently shake the plate to ensure mixing Place in a 37 C incubator for 10 minutes Add 25 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 Add the reconstituted FFA Reagent B to another disposable tray Add 50 of FFA Reagent B to each well Gently shake the plate to ensure mixing Place in a 37 C incubator for 10 minutes Allow the plate to equilibrate to room temperature for 5 minutes During this time ensure that there are no2. 00 Plate A to Plate B ECEE E 000000000000 0101010101010101001010 01010101010101010101010 01010107010701010101010 01010101010101010101010 07010107010101010101010 Reconstitute FFA Reagent A using Diluent A PRAA IAA ASA EEA React A Add 100 I well Incubate 10 minutes 37 C P AEA i 01010101010101010101010 50 I well Reconstitute FFA Reagent B using Diluent B 000000000000 FFA Reagent B Add 50 I well Incubate 10 minutes 37 C 000000000000 0100101010101010101010 01010101010101010101010 4 Place at room temp for 5 minutes Pop any bubbles in each well using a clean pipet tip or large gauge needle 4 Measure the optical density of each well at 540 nm using a spectrophotometer plate reader Plate C may be necessary for the assay of standards if al 96 wells of Plate A are used FREE GLYCEROL DETECTION One hour prior to assay reconstitute Glycerol Reagent A and prepare standards Keep all at room temp Remove 50 I well conditioned media oem 50 BGBGoSeOOOoD 01010101010101010101010 from Plate A to a blank assay plate Add OT IEEE IA TOE 50 I standards to empty wells 000000000000 000000000000 Add 50 I well reconstituted Glycerol Reagent A to 000000000000 GLYCEROL a blank assay plate including the glycerol PORR ROn REAGENT A standards at 50 well and optional plate without pibetctcenaetoeet cells Plate C may be necessary for the J assay of glycerol standards if al 96 wells of P
3. 063 0 019 4 1 0 05 0 006 1 4 0 046 0 002 o 0 044 0 y 0 0019x 0 0045 R 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 R value should be equal or greater then 0 98 for the standard curve to be valid Any R values below 0 98 must have the standard curve run again 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 OD Glycerol ee et Glycerol Standard Curve 0 50 100 150 200 250 uM Glycerol Intercept 0 0002 y 0 0009x 0 0002 R 0 999 0 1 e Series1 Linear Series1 OD Blank y observed O D minus the blank x 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 m x y 0 0075 0 003 where 0 003 slope of the line and 0 0075 y intercept Be careful to enter the proper 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 shou
4. BER YELLOW PINK YELLOW PINK ORANGE 40ML UNIT BOTTLE BOTTLE 1 ml VIAL 10 I VIAL 100 I VIAL 5OML 25ML BOTTLE BOTTLE 50 I VIAL BOTTLE Other equipment reagents required but not provided with the kit Blank 96 well plates Multi channel Pipet single channel pipet and pipet tips Plate reader with a filter of 540 nm Incubator at 37 C Large gauge needle Cultured human adipocytes Tubes for diluting glycerol standards QTY STORAGE 4 C 4 C 20 C 20 C 4 C ZG 4 C 4 C 4 C 20 C 4 C 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 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 H202 A quinoneimine dye is produced by the peroxidase catalyzed coupling of 4 aminoantipyrine 4 AAP and sodium N ethytl N 3 sulfopropyl m anisidine ESPA with H202 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 O DAP H20 H 0 4 AAP ESPA Quinone
5. Zenbio gt 96 well Adipocyte Lipolysis Assay Kit for Detection of Both Free Glycerol and Non Esterified Fatty Acids 500 point assay kit Cat LIP 3RB INSTRUCTION MANUAL 2ZBM0048 01 STORAGE CONDITIONS Reagents amp Buffers 4 C Vehicle amp Controls 20 C ALL ZEN BIO INC PRODUCTS ARE FOR RESEARCH USE ONLY NOT APPROVED FOR HUMAN OR VETERINARY USE OR FOR USE IN DIAGNOSTIC OR CLINICAL PROCEDURES LIMITED PRODUCT WARRANTY This 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 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 866 234 7673 Electronic mail e mail information zen bio com World Wide Web http www zenbio com 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 the bloodstream w
6. 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 The optical density of each well is then measured at 540 nm 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 I of Wash Buffer into the 1 mM glycerol standard tube provided and mix well by vortexing This produces a diluted stock glycerol standard of 200 M Pipette 125 Iof wash 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 the wash buffer serves as the zero standard 200 1 125 1 125 1125 1125 1125 1 125 1 Also at this time prepare the Glycerol Reagent A by adding 40 ml room temperature deionized water per bottle following the instructions on the bottle Gently invert bottle to mix contents DO NOT VORTEX Use a pipet to ensure that the powder is completely dissolved 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 At the end of the incubation an additional 50 of the conditioned media is removed and transferred to the
7. corresponding well of a blank plate for assessment of free glycerol This is most easily accomplished using a multi channel pipet Add 50 of each glycerol standard to any remaining empty wells in one of the blank assay plates OPTION to determine if the compound alone reacts with the Glycerol Reagent A prepare a fresh plate not included in kit containing 50 of the compound This plate can be incubated at 37 C with the treated cells When performing the assay add 50 of Glycerol Reagent A following the instructions in Steps 5 and 6 Add the reconstituted Glycerol Reagent A solution to one of the disposable trays provided in the kit Add 50 I of Reagent A to each well of Plate B and Plate C if used 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 25 C room temperature for 15 minutes The optical density of each well is then measured at 540 nm 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 std ee 333 0 68 0 636 111 0 244 a a 37 0 104 0 06 12 3 0
8. here 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 Alterations in lipolytic capacity have also been implicated in the susceptibility to obesity of African American 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 proteins in 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 rate limiting enzyme regulating adipocyte lipolysis then catalyzes 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 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 te
9. imine dye H20 Detection of Non Esterified Fatty Acids Free Fatty Acids FFA Assessment of lipolytic activity can also be detected 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 ACS HCOOH ATP CoA Acyl CoA AMP PP with oxygen in the presence of acyl CoA NEFA oxidase ACOD to produce hydrogen peroxide Hydrogen peroxide in the Acyl CoA o SCS 2 3 4rans Enoyl CoA H O presence of peroxidase POD allows the oxidative condensation of 3 methyl N sie CH ethyl N hydroxyethyl aniline with 4 ae a eee aminoantipyrine which forms a purple Y gt ara PODL Ease ne product that absorbs light at 550nm This te allows the concentration of NEFA to be determined from the optical density measured at 540 550nm N NOTE 3 fatty acid molecules are released per triglyceride molecule resulting in a 3 1 fatty acid to glycerol concentration 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
10. late A are used Incubate at 25 C room temperature for 15 minutes Pop the bubbles in each well 4 Measure the optical density of each well at 540 nm using a spectrophotometer plate reader REFERENCES Doo OT A oN a 8 9 Arner P 1996 Diabetes Rev 4 4 450 463 Botion LM amp Green A Diabetes 1999 48 1691 1697 Brasaemle DL Dolios G Shapiro L Wang R 2004 J Biol Chem 279 45 46835 42 Cooper DMF Schlegel W Lin MC Rodbell M 1979 J Biol Chem 254 18 8927 8931 Dyck DJ Can J Appl Physiol 2000 25 6 495 523 Kordik CP amp Reitz AB J Medicinal Chem 1999 42 2 181 201 Rieusset J Chambrier C Bouzakri K Dussere E Auwerx J Riou J P Laville M Vidal H Diabetologia 2001 44 544 554 Robidoux J Martin TL Collins S 2004 Ann Rev Chem 253 7570 7578 Scriba D Aprath Husmann 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
11. ld 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 R value should be equal or greater then 0 98 for the standard curve to be valid Any R 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 APPENDIX A PLATE LAYOUT APPENDIX B PROCEDURE FLOWCHART Remove 150 of the shipping medium and place in your incubator for 5 7 days 3 5 days for international customers 4 ON DAY OF ASSAY Make all test compound dilutions in Assay Buffer Plate A OOO0000000000 O000000000000 y O000000000000 Remove 120 media from all wells EA Add 200 Wash Buffer to all wells OOOO OO OOELN Plate A Remove 120 media amp Wash oeeo ee aaa Buffer Add another 200 Wash 000000000000 Buffer to all wells 000000000000 Plate A 000000000000 Remove all media amp Wash Buffer Add 100 eee OCCO treatments controls to 3 wells at a time otorefeyoleretelereie e 000000000000 000000000000 4 Incubate 3 5 hours at 37 C 4 120 media 200 Wash Buffer 200 Wash Buffer Add another 200 Wash Buffer Remove 3 wells at a time Add treatments 3 wells at a time FREE FATTY ACID DETECTION PES OO00000000000 Remove 30 l well conditioned media from 0000000000
12. st compounds affect lipolysis via adrenergic receptors 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 NOREPINEPHRINE Bas Bas _ ABBREVIATIONS AC adenylate cyclase AR adrenergic receptors G G protein coupled receptor S FFA free fatty acids PKA protein kinase AMP adenosine monophosphate ATP adenosine triphosphate 7 IR insulin receptor i PDE _ phosphodiesterase HSL Pe Be mc triglyceride oe TG HERS a ee ae ee ee ee ee ee eee wee eee cnet Cocevccce FFA glycerol bloodstream ITEMS INCLUDED IN THE KIT ITEM LIP 2 3 Assay Buffer Wash Buffer Vehicle Positive control FFA Standard FFA Diluent A FFA Diluent B FFA Reagent A FFA Reagent B Glycerol Standard Glycerol Reagent A DESCRIPTION 500 ml LIP 2 3 Wash Buffer 250 ml 0 1 DMSO in LIP 2 3 Assay Buffer Isoproterenol 10 mM in DMSO Dilute to 1_M in Assay Buffer before use i e 1 Iin 10 ml Assay Buffer 1mM Stock See page 5 for standard curve preparation Reconstitute using 50 ml FFA Diluent A Discard remainder after 10 days Reconstitute using 25 ml FFA Diluent B per bottle Discard remainder after 10 days Glycerol 1mM see page 6 for dilution instructions 40 ml Reconstitute with 40 ml deionized water prior to use PURPLE BLUE AM
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