Cultured Human Adipocyte Lipolysis Assay Kit - Combo (LIP
Contents
1. Av OD OD on FFA Standard Curve uM FFA OD OD blank blank blank 0 80 0 0 05 0 048 0 049 070 y 0 002x 0 001 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 0 50 12 3 0 070 0 075 0 021 0 026 0 024 S 0 40 37 0 119 0 122 0 070 0 073 0 072 a 0 30 111 0 274 0 277 0 225 0 228 0 227 ee 333 0 689 0 750 0 640 0 701 0 671 l 0 10 0 00 0 0 100 0 200 0 300 0 400 0 Slope 0 002 uM FFA Intercept 0 001 R 1 000 y observed O D 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 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 Rev 05 20 11 Page 9 of 14 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 OuM s2. Wash Buffer to make the 200 uM VIAL glycerol standard see page 6 for recommended dilution scheme LIP2 3 LIP2 3 Assay Buffer Buffer doom ml 100 ML ML ee essay Suter Standard see a See tage Seno Stock See page 5 for standard 100 on jt Curve preparation VIAL ee Diluent B PINK FFA Reagent A accanstiis Wang TOR A FER BIA using 10 5 ml FFA Diluent is BOTTLE Z A Discard remainder after 10 days FFA Reagent B Reconstitute using 5 5 ml FFA Diluent PINK BOTTLE 1 4 C B Discard remainder after 10 days Other equipment reagents required but not provided with the kit e Multi channel Pipet single channel pipet and pipet tips e Plate reader with a filter of 540 nm e Incubator at 37 C e Large gauge needle e 96 well plate of adipocytes LIP 3 cat SA 1096 e Tubes for dilution of standards Rev 05 20 11 Page 4 of 14 ASSAY PROCEDURE 1 Preadipocytes are plated in 96 well plates and allowed to differentiate under standard Zen Bio differentiation conditions for 1 week Upon arrival remove 150ul 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
3. amp Wash Buffer Add another 200 ul Wash Buffer to all wells Remove all media amp Wash Buffer Add 150 ul treatments controls to 3 wells at a time Incubate 3 5 hours at 37 C Plate A O00000000000 O00000000000 O00000000000 O00000000000 000000000000 Plate A O00000000000 O00000000000 O00000000000 O00000000000 000000000000 Plate A 000000000000 O00000000000 O00000000000 O00000000000 000000000000 120 ul media 200 ul Wash Buffer 200 ul Wash Buffer Add another 200 ul Wash Buffer Remove 3 wells at a time Add treatments 3 wells at a time FREE FATTY ACID DETECTION Plate A Remove 30 ul well conditioned media from 000000000000 O00000000000 Plate A to Plate B 000000000000 O00000000000 Reconstitute FFA Reagent A using Diluent A Add 100u well Incubate 10 minutes 37 C 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 Measure the optical density of each well at 540 nm using a spectrophotometer plate reader Rev 05 20 11 0 01000 0 010 0 010 0 0 010700 0 010 0 010 0 0 0 00 0 0 010 0 010 0 0 0 000 0 010 0 010 0 0 01000 0 010 0 010 0 0 0 000 0 010 0 010 0 0 010700 0 010 0 010 0 0 000 0 0 010 0 010 0 0 01000 0 010 0 010 0 0 0 000 0 010 0 010 0 Page 1
4. of 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 uM stock dilution serves as the highest standard and the wash buffer serves as the zero standard 400 pl 250 ul 250 pl 250 pl 250 pl 250 pl 250 pl Se S586 200 100 50 25 12 5 6 25 3 125 uM uM uM uM uM uM pM 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 2 Also at this time prepare the Glycerol Reagent A by adding 11 0 ml room temperature deionized water per bottle and gently invert DO NOT VORTEX Use a pipet to ensure that the powder is completely 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 Rev 05 20 11 Page 7 of 14 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 7 days refrigerated 2 8 C store any remaining solution refrigerated 2 8 C 3 At the end of the incubation an additional 100 ul of the conditioned media is removed and transferred to the corresponding well of a b
5. receptor GD FFA glycerol PDE _ phosphodiesterase E l ee 1G triglyceride FFA glycerol T bloodstream Rev 05 20 11 Page 2 of 14 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 gt G 1 P ADP G 1 P O2 gt DAP H20 H20 4 AAP ESPA gt Quinoneimine 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 with oxygen in t
6. 3 of 14 Assay Plate 30 ul 50pl well FFA Reagent B 0 010700 0 010 0 010 0 0 01000 0 010 0 010 0 0 0 000 0 010 0 010 0 0 010700 0 010 0 010 0 0 0 07000 010 0 010 0 100pl well FFA Reagent A 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 100 ul well conditioned media ee 100 pl 900000990099 0 0100 0 0 010 0 010 0 from Plate A to a blank assay plate Add CEET ETTE 000000000000 100 ul standards to empty wells 000000000000 000000000000 Add 100 ul well reconstituted Glycerol Reagent A to lolerejelerereleverere e GLYCEROL SRS a blank assay plate including the glycerol EAN pete REAGENT A eee H R 0 00 01010 01010 0 0 0 standards at 100uI well and optional plate without do00000 00000 RR cells Plate C may be necessary for the assay of glycerol standards if al 96 wells of Plate A are used Incubate at 25 C room temperature for 15 minutes Pop the bubbles in each well Measure the optical density of each well at 540 nm using a spectrophotometer plate reader REFERENCES 1 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 1
7. 979 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 8 Robidoux J Martin TL Collins S 2004 Ann Rev Chem 253 7570 7578 9 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 pe OF a Be ey NS Rev 05 20 11 Page 14 of 14
8. 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 as a vehicle control PLEASE NOTE ZEN BIO DOES NOT RECOMMEND THE USE OF SOLVENTS AT CONCENTRATIONS ABOVE 1 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 150 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 plate at 37 C humidified incubator for 3 hours for time course experiments the longest time point recommended is 5 hours Note Treatme
9. 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 B adrenergic agonist is used as the positive control if your test compounds affect lipolysis via B adrenergic receptors Robidoux et al 2004 This lipolysis assay kit provides the tool to study chemical compounds that may influence lipolysis in cultured human adipocytes EPINEPHRINE NOREPINEPHRINE By Bo Figure 1 Overview of adipocyte lipolysis ABBREVIATIONS AC adenylate cyclase AR adrenergic receptors S 5 Gs G protein coupled receptor i ATP ai FFA free fatty acids fi S AMP N PKA _ protein kinase PKA AMP adenosine monophosphate t S ATP adenosine triphosphate y EE IR insulin
10. ain Data are expressed as uM glycerol released OPTION express data as Fold induction over appropriate vehicle Fold induction uM glycerol SAMPLE uM glycerol VEHICLE Rev 05 20 11 Page 10 of 14 TROUBLESHOOTING Problem Suggestions High background or the glycerol reagent A turns purple before the assay begins e Change pipet tips frequently e Use Glycerol Reagent A before the expiration date e Make sure to starve the cells for 5 7 days BEFORE initiating treatment e Ensure a saturated humidity in the incubator to prevent Edge effects l evaporation from the outside wells e 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 No response to positive control Inconsistent OD reading FREQUENTLY ASKED QUESTIONS 1 Can I buy the reagents separately The Glycerol Standard cat LIP GLYSTAN Free fatty Acid Standard cat FFA STAN and Glycerol Reagent A cat RGTA 10 are sold separately LIP 2 3 Assay Buffer Free Fatty Acid Reagents and Diluents A and B are not sold separately 2 need to know the concentration of the BSA in the Assay Buffer ZenBio Inc does not provide the concentrations of the components of our media and buffers If knowledge of the BSA concentration is critical to your experiment you may order Assay Buffer WITHOUT BSA for no additional charge Please note it on y
11. erified fatty acids This is most easily accomplished using a multi channel pipet Add 30 ul of each standard to empty wells 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 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 Rev 05 20 11 Page 6 of 14 6 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 7 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 8 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 400 ul 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 uM Pipette 250 ul
12. he presence of acyl CoA oxidase ACS ACOD to produce hydrogen peroxide EEA ATP CoA Acyl CoA AMP PP Hydrogen peroxide in the presence of peroxidase POD allows the oxidative ACOD Acyl CoA O 2 3 trans Enoyl CoA H O CoH N N at C H OH ANS N O C H OH 4H O NOTE 3 fatty acid molecules are released per triglyceride molecule resulting in a 3 1 fatty acid to glycerol concentration condensation of 3 methyl N ethyl N hydroxyethyl aniline with 4 aminoantipyrine which forms a purple product that absorbs NH light at 550nm This allows the concentration N Ao N C H of NEFA to be determined from the optical 9 N density measured at 540 550nm Rev 05 20 11 Page 3 of 14 ITEMS INCLUDED IN THE KIT ITEM DESCRIPTION Cap UNIT QTY STORAGE Color Adipocytes Plate A Cultured human subcutaneous or PLATE 1 37 C omental adipocytes Assay Plates 96 well assay plate blank Se L E Wash Buffer Buffer SOML Vehicle 0 1 DMSO in LIP 2 Assay Buffer PURPLE 1 ml e e cal 4 Positive control Isoproterenol 10 mM in DMSO Dilute BLUE 10 pl Eaa to 1 uM in Assay Buffer before use VIAL i e 1 ulin 10 ml Assay Buffer Glycerol Reagent A Reconstitute with 11 0 ml deionized BOTTLE cat RGTA 10 water prior to use i rer For multi channel pipetters clear EACH e HI ad Kal Glycerol standard Glycerol 1mM Dilute with 400 ul ORANGE 100 ul Ban cat LIP GLYSTAN
13. lank plate for assessment of free glycerol This is most easily accomplished using a multi channel pipet Add 100 OI of each glycerol standard to any remaining empty wells in one of the blank assay plates 4 OPTION to determine if the compound alone reacts with the Glycerol Reagent A prepare a fresh plate not included in kit containing 100 ul of the compound This plate can be incubated at 37 C with the treated cells When performing the assay add 100 ul of Glycerol Reagent A following the instructions in Steps 5 and 6 5 Add the reconstituted Glycerol Reagent A solution to one of the disposable trays provided in the kit Add 100 ul 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 6 The optical density of each well is then measured at 540 nm Rev 05 20 11 Page 8 of 14 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
14. lephone 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 e World Wide Web http www zenbio com Rev 05 20 11 Page 1 of 14 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 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 is believed 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 fS agonists which activate B
15. nt times longer than 3 hours will result in significant fatty acid reutilization by the adipocytes and may decrease signal relative to total lipolysis activity Rev 05 20 11 Page 5 of 14 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 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 120 ul of Assay Buffer into 6 tubes not provided Pipette 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 60Oul 6Oul GOul 6Op GOpl 60p1 FFA 111 12 3 4 1 Std a uM a uM uM a 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 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 8 C At the end of the incubation 30 ul of the conditioned media is removed and transferred to the corresponding well of a blank plate for assessment of non est
16. our order 3 What is the Free fatty acid standard Free Fatty Acid standard cat FFA STAN is oleic acid in an aqueous buffer 4 have LIP 1 Assay Buffer leftover from another kit May I use it in this assay No The use of LIP 1 Assay Buffer with this kit will result in inconsistent data LIP 2 3 Assay Buffer contains components that are essentially free of interfering fatty acids whereas the LIP 1 Assay Buffer does not 5 have more samples plus standards to run than can fit on 1 96 well plate Can compare data obtained from multiple plates The lipolysis kit is designed for the assay of a single plate You may 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 remainder of your samples assayed In order to obtain comparable data both plates must be assayed on the same day using kits and cells from the same lot number An additional blank assay plate is provided for the assay of glycerol standards Rev 05 20 11 Page 11 of 14 APPENDIX A PLATE LAYOUT Rev 05 20 11 Page 12 of 14 APPENDIX B PROCEDURE FLOWCHART Remove 150ul of the shipping medium and place in your incubator for 5 7 days 3 5 davs for international customers 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
17. tandard from all OD values including the standard curve uM OD OD RA Glycerol Standard Curve glycerol OD OD blank blank blank 0 700 y 0 003x 0 001 0 0 044 0 041 0 043 3 125 0 054 0 053 0 012 0 011 0 011 6 25 0 062 0 063 0 020 0 021 0 020 0 500 12 5 0 083 0 084 0 041 0 042 0 041 oso 25 0 126 0 125 0 084 0 083 0 083 a 50 0 205 0 208 0 163 0 166 0 164 l 100 0 372 0 374 0 330 0 332 0 331 0 200 200 0 698 0 697 0 656 0 655 0 655 Slope 0 003 0 000 Intercept 0 001 0 50 100 150 200 250 R2 1 000 uM Glycerol y observed O D minus the blank x concentration of glycerol in uM 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 0 001 0 003 where 0 003 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 Any OD values greater than the highest standard 200 uM 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 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 ag
18. zenbio gt Cultured Human Adipocyte Lipolysis Assay Kit for Detection of Both Free Glycerol and Non Esterified Fatty Acids CAT LIP 3 LIP 3 OM LIP 3 NC INSTRUCTION MANUAL 2ZBM0011 08 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 Reagents amp Buffers 4 C Use reconstituted Glycerol Reagent A within 7 days 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 Te
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