(LIP-3-L1) - Zen
Contents
1. Plate A Plate A 200 ul Wash Buffer 200 ul Wash Buffer Assay Plate 120 ul media Add another 200 ul Remove 3 wells at a time Add treatments 3 wells at a time 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 i Measure the optical density of each well at 540 nm using a spectrophotometer plate reader REV 04 22 10 Page 13 of 14 FFA Reagent B 100ul well FFA Reagent A 50ul well Plate C may be necessary for2. N D CH POD N 2 2 oo di C H OH concentration of NEFA to be determined from the optical density measured at 540 550nm 3 fatty acid molecules are released per triglyceride molecule resulting in a 3 1 fatty acid to glycerol concentration REV 04 22 10 Page 3 of 14 ITEMS INCLUDED IN THE KIT ITEM DESCRIPTION Cap UNIT QTY STORAGE Color Plate A 96 well plate 3T3 L1 preadipocytes PLATE 37 C LIP 1 L1 ONLY Assay Plates Plates 96 well 96 wellassayplate blank 96 wellassayplate blank blank PLATE Medium 313 bn cin rene cat PM 1 L1 E Differentiation Medium na Adipocyte SERIO Medium cat BOTTLE Adipocyte Medium 3T3 L1 Arsen _ cat BOTTLE Wash Buffer Buffer 50M Vehicle 0 196 DMSO in LIP 2 3 Assay Buffer PURPLE 1 ml 20 ES Positive control Isoproterenol 10 mM in DMSO Dilute to 1 uM BLUE 10 ul 20 C in Assay Buffer before use 1 9 1 ul in 10 ml VIAL aL ined Glycerol Reagent A Reconstitute with 11 0 ml deionized water prior BOTTLE T un i M T LLL days Glycerol standard Glycerol 1mM Dilute with 400 ul Wash ORANGE 100 ul 20 C Buffer to make the 200 uM glycerol standard VIAL see page 8 for recommended dilution scheme E LIP2 3 LIP2 3 Assay Buffer Buffer 100m 100m ef fOw 4 FFA 1mM ME See page 7 for standard curve AMBER 100 ul C Eu E B FFA Diluent A A YELLOW
3. M FFA Diluent B B a 5 5ML 5ML eme e Discard remainder after 10 days o 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 Sterile trays for multi channel pipetters during differentiation of cells Plate reader with a filter of 540 nm Incubator at 37 C Large gauge needle Additional 96 well plate of adipocytes cat SA 1096 Tubes for dilution of standards REV 04 22 10 Page 4 of 14 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 3T3 L1 Differentiation Medium cat DM 2 L1 see table 1 below for recommended volumes Incubate for 3 days 5 Remove the ST3 L1 Differentiation Medium and replace with 3T3 L1 Adipocyte Maintenance Medium Incubate for 2 3 days 6 Feed cells every 2 3 days using 3T3 L1 Adipocyte Maintenance Medium until ready for assa
4. not included in kit containing 50 ul of the compound This plate can be incubated at 37 C with the treated cells When performing the assay add 50 ul of Glycerol Reagent A following the instructions in oteps 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 room temperature for 15 minutes 6 The optical density of each well is then measured at 540 nm 04 22 10 Page 9 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 Avg OD OD OD FFA Standard Curve uM FFA OD OD blank blank blank 005 0048 9049 12 3 37 0 072 y 0 002 0 001 R 1 000 series iniear Series ox E e o 0 0 1000 2000 300 0 400 0 uM FFA Re 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
5. 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 p agonists which activate p 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 trig
6. Auwerx J Riou J P Laville M Vidal H Diabetologia 2001 44 544 554 8 Robidoux J Martin TL Collins S 2004 Ann Hev 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 UBMB Life 56 7 379 85 REV 04 22 10 Page 14 of 14
7. 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 value should be equal or greater then 0 98 for the standard curve to be valid Any values below 0 98 must have the standard curve run again 04 22 10 Page 10 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 standard from all OD values including the standard curve Avg Z glycerol OD OD blank blank blank GUT yz20003x 0 004 0 044 0 041 0 043 0 011 12 5 0 042 25 50 0 164 OD Blank Series Linear Series 0 655 1 150 250 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 ent
8. ON 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 Dilution Buffer alone serves as the zero standard 60 1 Oul Oul Oul Oul 60 111 123 4 1 Std 2 uM HM pM M 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 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 3 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 esterified fatty acids This is most easily accomplished using a multi channel pipet Add 30 ul of each standard to empty wells 4 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 ensur
9. e mixing Place in a 37 C incubator for 10 minutes 5 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 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 04 22 10 Page 7 of 14 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 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 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 serve
10. er 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 value should be equal or greater then 0 98 for the standard curve to be valid Any values below 0 98 must have the standard curve run again 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 04 22 10 Page 11 of 14 APPENDIX A PLATE LAYOUT REV 04 22 10 Page 12 of 14 APPENDIX B 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 i Remove 120 ul media amp Wash Buffer Add another 200 ul Wash Buffer to all wells i Remove all media amp Wash Buffer Add 150 ul treatments controls to 3 wells at a time i Incubate 3 5 hours at 37 C i FREE FATTY ACID DETECTION Remove 30 ul well conditioned media from Plate A to Plate B Add 30 ul FFA standards to empty wells Plate A Plate A
11. lycerides 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 Figure 1 Overview of adipocyte lipolysis EPINEPHRINE NOREPINEPHRINE D4 d Bos ABBREVIATIONS AC adenylate cyclase AR adrenergic receptors MC G protein coupled receptor FFA free fatty acids PKA protein kinase AMP adenosine monophosphate DE E ATP adenosine triphosphate Y T IR insulin receptor Vs H E PDE phosphodiesterase p TG FFA glyc 4 TG triglyceride FFA glycerol aa bloodstream REV 04 22 10 Page 2 of 14 PRINCIPLES OF 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 gl
12. s as the zero standard 400 pl 250 0 250 250 ul 250 ul 250 ul 250 ul 200 100 50 25 12 5 6 25 3 125 uM uM uM uM uM uM 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 eight 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 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 blank plate for assessment of free glycerol This is most easily accomplished using a multi channel pipet Add 100 ul of each glycerol standard to any remaining empty wells in one of the blank assay plates REV 04 22 10 Page 8 of 14 4 OPTION to determine if the compound alone reacts with the Glycerol Reagent A prepare a fresh plate
13. 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 i Remove 100 ul well conditioned media oooooooooooo 100 ul from Plate A to a blank assay plate Add 000000000000 900000000000 100 ul glycerol standards to empty wells Add 100 ul well reconstituted Glycerol Reagent A to 000000000000 000 a blank assay plate including the glycerol i 0100 standards at 50ul well and optional plate without 000000000000 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 2 Botion LM amp 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 amp Reitz AB J Medicinal Chem 1999 42 2 181 201 7 Rieusset J Chambrier C Bouzakri K Dussere E
14. tions 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 media 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 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 4 Incubate the plate 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 04 22 10 Page 6 of 14 A DETECTION OF NON ESTERIFIED FATTY ACIDS 1 Prepare the standard curve using the FFA STANDARD SOLUTI
15. y 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 L1to Change AM 1 L1 to mL SLM DM 2 L1 AM 1 L1 AM 1 L1 OUT IN OUT IN OUT OUT _ 96 plate 150ul well 150 well 90 ul well 120ul well 90 ul well 1204 well 48 well plate 300 ul well 300 ul well 200 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 7 9 11 13 15 oroliferation gt lt 48 hrs Feed Feed Feed 10096 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 f nucleus Once the cells are 10096 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 04 22 10 Page 5 of 14 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 LIP 2 3 Assay Buffer 100 ml is available NOTE if desired maintain a constant concentration of solvent by preparing all compound dilu
16. ycerol 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 H203 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 O DAP HO H20 4 AAP ESPA Quinoneimine dye H O 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 the presence of acyl CoA oxidase ACOD to produce MTS ATP CoA AoyeCoA AMP PP hydrogen peroxide Hydrogen peroxide in the presence of peroxidase POD allows the ACOD oxidative condensation of 3 methyl N ethyl N LC B hydroxyethyl aniline with 4 aminoantipyrine which forms a purple product NH that absorbs light at 550nm This allows the E lat 2
17. zenbio P Lipolysis Assay Kit for 3T3 L1 Cells Detection of Both Free Glycerol and Non Esterified Fatty Acids CAT LIP 3 L1 LIP 3 NC L 1 INSTRUCTION MANUAL 42 09 STORAGE CONDITIONS 96 well plate cultured 3T3 L1 preadipocytes LIP 3 L1 37 C incubator e Reagents amp Buffers 4 C e 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 e Zen Bio Inc 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 1 866 ADIPOSE 866 234 7673 e Electronic mail e mail information zen bio com e World Wide Web http www zenbio com REV 04 22 10 Page 1 of 14 INTRODUCTION Lipolysis plays a central role in the regulation of energy balance Lipolysis is the process in which triglycerides
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