Cultured Human Adipocyte Lipolysis Assay Kit
Contents
1. 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 blank plate GLYCEROL REAGENT A An additional blank assay plate may be necessary for the assay of glycerol standards if al 96 wells are used2. PKA protein kinase ji 1 AMP adenosine monophosphate PA A ATP adenosine triphosphate PKA s IR insulin receptor v ne PDE phosphodiesterase P ne TG triglyceride FFA glycerol bloodstream Rev APRIL 19 2010 Page 2 of 10 PRINCIPLE OF THE ASSAY 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 quinoeimine dye is produced by the peroxidase catalyzed coupling of 4 aminoantipyrine 4 AAP and sodium N ethyl 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 O0 DAP H20 H20 4 AAP ESPA gt uinoneimine dye H20 ITEMS INCLUDED IN THE KIT ITEM DESCRIPTION Cap UNIT QTY STORAGE Color Adipocytes Plate A Cultured human subcutaneous adipocytes Plate 1 37 Blank Assay Plates 96 well assay plates blank Oo PLATE 2 Assay Buffer 100M ooo wu ME 5 ROE Wash Buffer Buffer SM ooo ml BOTTLE VIAL Positive control Isoproterenol 10 mM in DMSO Dilute to 1 uM in BLUE 10 pl 20 Assay Buff
3. 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 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 bufter 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 or optionally IBMX for treatments 5 24 hours 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 shou
4. 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 1 866 ADIPOSE 866 234 7673 e Electronic mail e mail information zen bio com e World Wide Web http www zenbio com Rev APRIL 19 2010 Page 1 of 10 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 a 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 regu
5. Remove 120 ul media from all wells Add 200 ul Wash Buffer to all wells Remove 200 ul media 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 OPTION Add 100 ul well compounds to a fresh plate without cells Incubate 3 5 hours at 37 C One hour prior to assay reconstitute Glycerol Reagent A and prepare standards Keep all at room temp Remove 100 ul well conditioned media from Plate A to a blank assay plate Add 100 ul well glycerol standards to empty wells Add 100 ul well reconstituted Glycerol Reagent A to the plate including the glycerol standards at 100ul well and optional plate without cells 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 Rev APRIL 19 2010 Page 10 of 10 Plate A Plate A Plate A Plate A
6. 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 uM OD OD an nena ndard Curve glycerol PRE blank blank o NI NAM 0 088 OD Blank 0 188 0 382 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 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 APRIL 19 2010 Page 6 of 10 TROUBLESHOOTING Problem Suggestions High background or the glycerol reagent A e Change pipet tips frequently turns purple before the assay
7. begins Use Glycerol Reagent A before the expiration date No response to positive control e Make sure to starve the cells for 5 7 days BEFORE initiating treatment DO NOT use IBMX as the positive control if you are incubating for less than 5 hours Edge effects Ensure a saturated humidity in the incubator to prevent evaporation from the outside wells Inconsistent OD reading 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 FREQUENTLY ASKED QUESTIONS 1 want to perform a lipolysis time course experiment How many time points can complete We do not recommend performing more than 2 time points per assay For time course experiments add 250 ul assay medium with treatments per well Remove 100 ul for each time point 2 When do need to use the IBMX positive control you use the 3 5 hour incubation described in this manual you will not need to use the IBMX as your positive control The IBMX positive control is designed for treatments ranging from 5 24 hours The IBMX alternate control may be used in addition to the Isoproterenol positive control if your treatment time will exceed 5 hours 3 Can buy the reagents separately The Glycerol Standard cat LIP GLYSTAN and Glycerol Reagent A cat RGTA 10 are sold separately Assay Buffer is not sold separately 4 need to know t
8. er before use 1 6 1 ul in 10 ml Assay VIAL Buffer Glycerol Reagent A pogo ET with 11 0 ml deionized water prior to ere Tray Ki ees channel For multi channel pipetters clear polyvinyl clear For multi channel pipetters clear polyvinyl EACH ane standard Glycerol 1mM Reconstitute with 400 ul Wash nm 100 hol Buffer to make the 200 uM glycerol standard see VIAL page 5 for recommended dilution scheme ALTERNATE 3 lsobutyl 1 methylxanthine IBMX 100 mM in 10 20 ial Positive control DMSO Dilute to 100 uM in Assay Buffer before VIAL use i e 1 ulin 1 ml Assay Buffer USE ONLY IF YOUR TREATMENT TIME EXCEEDS 5 HOURS Other equipment reagents required but not provided with the kit Multi channel Pipet single channel pipet and pipet tips Plate reader with a filter of 540 nm Incubator at 37 C Large gauge needle Option Step 5 of Assay Procedure 96 well plate blank Tubes for dilution of standards Rev APRIL 19 2010 Page 3 of 10 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
9. he 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 your order 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 Rev APRIL 19 2010 Page 7 of 10 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 6 do not have time to pop the bubbles and read the plate Can freeze the conditioned media in one of the assay plates provided with the kit How long can store the samples Yes The conditioned media can be immediately stored at 80 C for a maximum of 7 days Bring the conditioned media in the plate to room temperature BEFORE adding the Glycerol Reagent A and completing the assay 7 1 need to use another plate format than the 96 well Do you have a kit can use Yes We offer a lipolysis assay ki
10. ht fewer data points can be assayed with this kit 8 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 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 9 At the end of the incubation 100 ul of the conditioned media is removed and transferred to the corresponding well of Plate B 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 10 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 the assay plates containing samples 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 11 The optical density of each well is then measured at 540 nm Rev APRIL 19 2010 Page 5 of 10 GLYCEROL STANDARD CURVE Generate standard curve see
11. lation of lipid mobilization The main lipolytic pathway involves beta agonists B agonists which activate B 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 Figure 1 Overview of adipocyte lipolysis tin ABBREVIATIONS ACN tli AC adenylate cyclase EE 8 JAR adrenergic receptors aw ee Gs G protein coupled receptor Pse pd FFA free fatty acids a
12. ld be performed in triplicate 5 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 10 and 11 6 Incubate the plates at 37 C humidified incubator for 3 hours for time course experiments the longest time point is usually 24 hours 7 One hour prior to the assay prepare the glycerol standards as follows Rev APRIL 19 2010 Page 4 of 10 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 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 ul 250 ul 250 wl 250 ul 250 ul 250 ul 250 200 100 12 5 6 25 3 125 eo 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 eig
13. t containing a vial of subcutaneous preadipocytes media to differentiate the cells and the reagents to complete the lipolysis assay Catalog LIP 1 SF is suitable for 384 96 24 or 12 well plate format You must then transfer the conditioned assay buffer to a 96 well format to complete the assay 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 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 I 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 Londos C 2004 IUBMB Life 56 7 379 85 Rev APRIL 19 2010 Page 8 of 10 APPENDIX A PLATE LAYOUT esses Rev APRIL 19 2010 Page 9 of 10 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 ON DAY OF ASSAY Make all test compound dilutions in Assay Butter
14. zenbio Cultured Human Adipocyte Lipolysis Assay Kit Cat LIP 1 LIP 1 NC INSTRUCTION MANUAL ZBM0002 05 STORAGE CONDITIONS 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 Assay Plate A Cultured human adipocytes LIP 1 kit ONLY 37 C CO incubator Glycerol Reagent A amp Buffers Store at 4 C Glycerol Standards amp Controls store at 20 C Long term storage LIP 1 NC Reagents Only kit remove the glycerol reagent A from the box and place at 4 C store the rest of the kit at 20 C Reagents are good for 6 months if stored properly 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
Download Pdf Manuals
Related Search