Lipolysis Assay Kit for 3T3-L1 Cells Non
Contents
1. Add another 200 ul Wash Buffer Plate A OOOOOOOOOOOO ONO OOO Remove 3 wells at a time Add treatments 3 wells at a time Plate A blank plate Page 9 of 9 100ul well FFA Reagent A 50 Reagent An additional plate may be necessary for the assay of standards2. 500ul well 500 ul well 300 ul well 400 ul well 300 ul well 400 ul well 3T3 L1 Growth and Differentiation Feeding Schedule DAY DAY DAY DAY DAY DAY DAY DAY DAY 2 0 3 5 g 9 11 13 15 proliferation gt 8 hrs Feed Feed Feed 100 Feed Feed Feed Feed Feed Feed Feed PM 1 L1 PM 1 L1 PM 1 L1 confluent DM 2 L1 AM 1 L1 AM 1 L1 AM 1 L1 AM 1 L1 AM 1 L1 AM 1 L1 PREADIPOCYTE nucleus Once the cells are 100 confluent incubate an additional 48 hours before initiating differentiation MATURE ADIPOCYTE ets 313 L1 adipocytes are suitable for most assays 7 14 days post differentiation Rev 8 15 2008 Page 4 of 9 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 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 as a vehicle control PLEASE NOTE ZEN BIO DOES NOT RECOMMEND THE USE OF SOLVENTS AT CONCENTRATIONS ABOVE 1 2 Remove 120 ul medium from each well Gently add 200 ul Wash Buffer to all wells Remove 200 ul of the me
3. 8 15 2008 Page 5 of 9 30 304 30 30 30u 30 FFA 111 123 4 1 Std te uM je uM U 6 Add 10 5ml 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 2 850 7 At the end of the incubation 50 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 8 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 9 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 10 Add the reconstituted FFA Reagent B to the other disposable tray provided in 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 11 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 resu
4. FFA Reagents A and B and completing the assay Rev 8 15 2008 Page 7 of 9 APPENDIX A PLATE LAYOUT _ _ Rev 8 15 2008 Page 8 of 9 APPENDIX LIP 2 L1 PROCEDURE FLOWCHART ON DAY OF ASSAY Make all test compound dilutions in Assay Buffer 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 I Remove all media amp Wash Buffer Add 100 ul treatments controls to 3 wells at a time I Incubate 3 5 hours at 37 C l Remove 50 ul well conditioned media from Plate A to one of the blank assay plates provided Add 50 ul FFA standards to empty wells I Reconstitute FFA Reagent A using Diluent A Add 100ul well Incubate 10 minutes 37 C I Reconstitute FFA Reagent B using Diluent B Add 50ul well Incubate 10 minutes 37 C I 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 8 15 2008 Plate A plate of mature 3T3 L1 adipocytes Plate A 120 ul media 200 ul Wash Buffer PI A 200 ul Wash Buffer
5. allows the concentration of NEFA to be Pee ae C H OH determined from the optical density measured 5 g an at 540 550nm a ITEMS INCLUDED IN THE KIT ITEM DESCRIPTION Plate A 96 well plate 3T3 L1 preadipocytes Assay Plate 96 well assay plate blank for samples 8 standards Preadipocyte 313 L1 Preadipocyte Medium cat PM 1 L1 50ml Medium LIP 2 L1 ONLY a Differentiation 3T3 L1 Adipocyte Differentiation Medium cat DM 2 Medium L1 15ml LIP 2 L1 ONLY Adipocyte Medium 3T3 L1 Adipocyte Maintenance Medium cat AM 1 L1 100ml LIP 2 L1 ONLY LIP 2 3 Assay Buffer 100 ml Wash Buffer Vehicle 0 1 DMSO in LIP 2 3 Assay Buffer 1 mI VIAL Positive control Isoproterenol 10 mM in DMSO Dilute to 1 uM_in Assay BLUE 10 ul Buffer before use i e 1 ul in 10 ml Assay Buffer VIAL FFA Standard 1mM Stock See page 5 for standard curve preparation 100 ul VIAL FFADIuentA tw TOS FFADiuentB OOOO OOOO O OoOo l S FFA Reagent A Reconstitute using 10 5 ml FFA Diluent A Discard YELLOW BOTTLE P Ki FFA Reagent B Reconstitute using 5 5 ml FFA Diluent B Discard PINK BOTTLE remainder after 10 days Tray Other equipment reagents required but not provided with the kit e 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 37 C Large gauge needl
6. ay 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 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 B adrenergic agonist is used as the positive control if your test compou
7. dia and Wash Buffer from each well and replace with another 200 ul Wash Buffer 3 Remove all the media and Wash Buffer from the cells from triplicate wells Treat the cells with 100 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 4 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 5 Prepare the standard curve using the 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 uM fatty acid Prepare as follows The kit standard solution is the 1 0 mM standard Pipette 60 ul of Dilution Buffer into 6 tubes not provided Pipette 30 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 Dilution Buffer alone serves as the zero standard Rev
8. e e Tubes for dilution of standards Rev 8 15 2008 Page 3 of 9 ASSAY PROCEDURE A DIFFERENTIATION PROCEDURE 1 Preadipocytes are plated sub confluent in 3T3 L1 Preadipocyte Medium cat PM 1 L1 and shipped the next 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 day with 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 and replace with an appropriate volume 313 L1 Differentiation Medium cat DM 2 L1 see table 1 below for recommended volumes Incubate for 3 days 5 Remove the 3T3 L1 Differentiation Medium and replace with 313 L1 Adipocyte Maintenance Medium Incubate for 2 3 days 6 Feed cells every 2 3 days using 313 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 Growth and Differentiation Feeding Schedule Table 1 Feeding Volumes Change PM 1 L1 to Change PM 1 L1 to Change DM 2 L1 to Change AM 1 L1 to PM 1 L1 DM 2 L1 AM 1 L1 1 1 UT OUT OUT N 96 well plate 90ul well 90ul well 150ul well 150 ul well 90 ul well 120 well 90 ul well 120 well 48 well plate 300 ul well 300 ul well
9. lt in inaccurate absorbance readings 12 The optical density of each well is then measured at 540 nm Rev 8 15 2008 Page 6 of 9 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 uMstd OD OD zero Standard Curve 333 0 68 0 636 111 0 244 0 2 3 0 104 0 06 12 3 0 063 0 019 4 1 0 05 0 006 1 4 0 046 0 002 0 0 044 0 y 0 0019x 0 0045 R 0 9995 200 uM standard Data are expressed as uM free fatty acids released 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 do 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
10. nds 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 sm Ba AR Mies ABBREVIATIONS AC adenylate cyclase AR adrenergic receptors G protein coupled receptor os FFA free fatty acids cAMP U PKA _ protein kinase i 3 AMP adenosine monophosphate 3 SAME a z ATP adenosine triphosphate PKA 33 IR insulin receptor i PDE _ phosphodiesterase Pod 2 triglyceride FFA glycerol FFA glycerol ee bloodstream Rev 8 15 2008 Page 2 of 9 PRINCIPLE OF 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 ACS presence of acyl CoA oxidase ACOD to ee Acyl CoA AMP PP produce hydrogen peroxide Hydrogen NEFA peroxide in the presence of peroxidase POD allows the oxidative condensation of 3 Acy CoA 0 550 o3 trans Enoy CoA methyl N ethyl N B hydroxyethyl aniline with 4 aminoantipyrine which forms a purple product that absorbs light at 550nm This NH ie
11. zenbic Lipolysis Assay Kit for 3T3 L1 Cells Non Esterified Fatty Acids Detection 100 point assay kit Cat LIP 2 L1 LIP 2 NC L1 INSTRUCTION MANUAL 2ZBM0041 00 STORAGE CONDITIONS 96 well plate cultured 3T3 L1 preadipocytes LIP 2 L1 37 C incubator e Reagents amp Buffers 4 C e Vehicle amp Controls 20 C For in vitro Use Only 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 e 9200 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 1 866 ADIPOSE 866 234 7673 e Electronic mail e mail information zen bio com World Wide Web http www zenbio com Rev 8 15 2008 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 m
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