Cultured Human Adipocyte Lipolysis Assay Kit - NEFA - Zen
Contents
1. peroxide in the presence of peroxidase POD allows the oxidative condensation of 3 ATOA 3 05 ACOD 55 tans Enoyk CoA H O methyl N ethyl N B hydroxyethyl aniline with 4 aminoantipyrine which forms a purple product that absorbs light at 550nm This NH Tat allows the concentration of NEFA to be Ac a ise C H OH determined from the optical density measured J a DM D a 4H 0 at 540 550nm e arm e ITEMS INCLUDED IN THE KIT ITEM DESCRIPTION Cap UNIT QTY STORAGE Color Adipocytes Plate Cultured human subcutaneous adipocytes LIP 2 PLATE 1 37 C A only Assay Plate 96 well assay plate blank for samples amp standards PLATE 2 LIP 2 3 Assay 100 ml BOTTLE 1 4 C Buffer Wash Buffer 50 ml BOTTLE 1 4 C Vehicle 0 196 DMSO in LIP 2 3 Assay Buffer PURPLE 1ml 1 20 C VIAL Positive control Isoproterenol 10 mM in DMSO Dilute to 1 uM in BLUE 10 ul 1 20 C Assay Buffer before use i e 1 ul in 10 ml Assay VIAL Buffer FFA Standard 1mM Stock See page 5 for standard curve AMBER 100 ul 1 4 C preparation VIAL FFA Diluent A LABEL 10 5ML 1 4 C YELLOW FFA Diluent B LABEL PINK 5 5ML 1 4 C FFA Reagent A Reconstitute using 10 5 ml FFA Diluent A Discard LABEL BOTTLE 1 4 C remainder after 10 days YELLOW FFA Reagent B Reconstitute using 5 5 ml FFA Diluent B Discard LABEL PINK BOTTLE 1 4 C remainder after 10 days Tray For multi channel pipetters clear pol2. as a vehicle control PLEASE NOTE ZEN BIO DOES NOT RECOMMEND THE USE OF SOLVENTS AT CONCENTRATIONS ABOVE 196 3 Remove 120 ul medium from each well Gently add 200 ul Wash Buffer to all wells Remove 200 ul of the media and Wash Buffer from each well and replace with another 200 ul Wash Buffer 4 Remove all the media and Wash Buffer from the cells from triplicate wells Treat the cells with 75 ul of the test compounds resuspended in Assay Buffer three 3 wells at a time Treat with the 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 your test compounds are not dissolved in DMSO The assay should be performed in triplicate 5 Incubate the plates at 37 C humidified incubator for 3 hours for time course experiments the longest 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 to total lipolysis activity 6 Prepare the standard curve using the STANDARD SOLUTION as follows Briefly spin down the contents of the free fatty acid standard tube Standards are 0 1 4 4 1 12 3 37 111 and 333 uM fatty acid Prepare as follows Rev 11 30 2009 Page 4 of 9 The kit standard solution is the 1 0 mM standard Pipette 120 ul of Assay Buffer into 6 tubes not provided Pipette
3. minus the blank x concentration of FFA in uM To calculate x for each y i e to change the observed O D into FFA concentration use the following equation y slope times x plus intercept y mx b so x y b m x y 0 001 0 002 where 0 002 slope of the line and 0 001 y intercept Be careful to enter the proper sign for the y intercept value as it may be a negative number Data are expressed as uM free fatty acids released Rev 11 30 2009 Page 6 of 9 OPTION express data as Fold induction over appropriate vehicle Fold induction uM free fatty acids SAMPLE uM 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 FREQUENTLY ASKED QUESTIONS 1 I do not have time to run the assay Can freeze the conditioned media in PLATE B How long can store the samples before 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 FFA Reagents A and B and completing the assay Rev 11 30 2009 Page 7 of 9 Page 8 of 9 Rev 11 30 2009 APPENDIX B LIP 2 PROCEDURE FLOWCHART Remove 150u of the shipping medium and place in your incubator for 5 7 days 3 5 days for international customers ON DAY OF ASSAY Make all test compound dilutions in Assay Buffe
4. 6 ADIPOSE 866 234 7673 e Electronic mail e mail information zen bio com e World Wide Web http Awww zenbio com Rev 11 30 2009 Page 1 of 9 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 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 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 f agonists which activate B adrenergic receptors via the intracellular G 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 prim
5. 60 ul 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 60001 60pl 60g 60l 60pl 60pl Note The above dilution series generates enough volume to perform the standard curve in duplicate If you wish to perform the standard curve in duplicate please note that seven fewer data points can be assayed with this kit 7 Add 10 5 ml 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 8 At the end of the incubation 50 ul 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 50 ul of each standard to empty wells 9 Add the reconstituted FFA Reagent A to one of the disposable trays provided in the kit Add 100 ul of FFA Reagent A to each well Gently shake the plate to ensure mixing Place in a 37 C incubator for 10 minutes 10 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 Add the reconstituted FFA Reagent B to the other disposable tray provided in
6. e 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 B adrenergic agonist is used as the positive control if your test compounds affect lipolysis via B 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 ABBREVIATIONS i AC adenylate cyclase AR adrenergic receptors Gs G protein coupled receptor FFA free fatty acids PKA protein kinase i AMP adenosine monophosphate i ATP adenosine triphosphate IR insulin receptor i PDE _ phosphodiesterase TG triglyceride ee ee FFA glycerol bloodstream Rev 11 30 2009 Page 2 of 9 PRINCIPLE OF THE ASSAY Assessment of lipolytic activity is 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 with oxygen in the presence of acyl CoA oxidase ACOD to OORA UE ACS AcykCoA AMP PP produce hydrogen peroxide Hydrogen NEFA
7. r Remove 120 ul media from all wells Add 200 ul Wash Buffer to all wells Remove 120 ul media amp Wash Buffer Add another 200 ul Wash Buffer to all wells Remove all media amp Wash Buffer Add 75 ul treatments controls to 3 wells at a time Incubate 3 5 hours at 37 C Remove 50 yl well conditioned media from Plate A to one of the blank assay plates provided Reconstitute FFA Reagent A using Diluent A Add 100 l well Incubate 10 minutes 37 C l Reconstitute FFA Reagent B using Diluent B Add 50ul well Incubate 10 minutes 37 C Place at room temp for 5 minutes Pop any bubbles in each well using a clean pipet tip or large gauge needle l Measure the optical density of each well at 540 nm using a spectrophotometer plate reader Rev 11 80 2009 Page 9 of 9 Plate A 000000000000 000000000000 000000000000 000000000000 000000000000 Plate A 000000000000 000000000000 000000000000 000000000000 000000000000 Plate A 000000000000 000000000000 000000000000 000000000000 000000000000 Plate A 000000000000 000000000000 000000000000 000000000000 000000000000 o 0 0 0 0 00 0 000 0 o 0 0 0 0 000 000 0 0 0 0 0 0 00 0 0 00 0 0 0 0 0 0 0 0 0 000 0 0 0 0 0 0 00 0 0 09 0 0 0 0 0 0 0 0 0 0 00 0 o 0 0 0 0 0 0 0 0 00 0 o 0 0 0 0 00 0 009 0 o 0 0 0 0 00 0 0 09 0 0 0 0 0 0 0 0 0 0 09 0 120 ul media 200 ul Wash Buffer 200
8. the kit Add 50 ul of FFA Reagent B to each well Gently shake the plate to ensure mixing Place in a 37 C incubator for 10 minutes Rev 11 30 2009 Page 5 of 9 12 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 13 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 OuM standard from all OD values including the standard curve Note 1mM standard is commonly omitted from analysis due to lack of linearity between 333 uM and 1mM Optionally a 4 parameter fit may be used to calculate standard curve Avg OD OD OD FFA Standard Curve uM FFA OD OD blank blank blank 0 80 0 0 05 0 048 0 049 0 70 ae 1 4 0 051 0 053 0 002 0 004 0 003 0 60 4 1 0 056 0 058 0 007 0 009 0 008 E 0 50 123 0 070 0 075 0 021 0 026 0 024 s ert 37 0 119 0 122 0 070 0 073 0 072 vs 111 0 274 0 277 0 225 0 228 0 227 ER 333 0 689 0 750 0 640 0 701 0 671 0 00 0 0 100 0 200 0 3000 4000 uM FFA Slope 0 002 Intercept 0 001 R 1 000 y observed O D
9. ul Wash Buffer Add another 200 ul Wash Buffer Remove 3 wells at a time Add treatments 3 wells at a time 50 ul 100 well FFA Reagent A blank plate 0 0 0 0 0 0 0 0 009 0 0 0 0 0 0 0 0 0 0 09 0 0 0 0 0 0 0 0 0 009 0 0 0 0 0 0 00 0 0 09 0 0 0 0 0 0 0 0 0 0 09 0 50pl well FFA Reagent B An additional plate may be necessary for the assay of standards
10. yvinyl CLEAR EACH 2 e m Other equipment reagents required but not provided with the kit e Multi channel Pipet single channel pipet and pipet tips Plate reader with a filter of 540 nm Incubator at 37 C Large gauge needle Tubes for dilution of standards Rev 11 30 2009 Page 3 of 9 ioris PROCEDURE Preadipocytes are plated in 96 well plates and allowed to differentiate under standard Zen Bio differentiation conditions for 1 week Upon arrival remove 150yl of the shipping medium from each well and discard Place the plate Plate A in your incubator for 5 7 days 3 5 days for international 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 the cells under a microscope prior to performing the assay see the photograph in the Certificate of Analysis 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
11. zenbio y Cultured Human Adipocyte Lipolysis Assay Kit Non Esterified Fatty Acids Detection 100 point assay kit Cat LIP 2 LIP 2 NC INSTRUCTION MANUAL ZBM0010 04 STORAGE CONDITIONS e Human 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 e Reagents amp Buffers 4 C e Vehicle amp Controls 20 C e Assay plate A 96 well cultured human adipocytes 37 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 e Zen Bio Inc e 3200 Chapel Hill Nelson Blvd Suite 104 e PO Box 13888 e Research Triangle Park NC 27709 e Telephone 919 547 0692 e Facsimile FAX 919 547 0693 e Toll Free 1 86
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