Cellulite Treatment Screening Lipolysis Kit - Glycerol - Zen
Contents
1. Orlandi C Zechmeister D Botanical extracts used in the treatment of cellulite Dermatol Surg 2005 31 866 872 Huber C Meyer MS Schreier T Topical treatments for Orange Peel Skin Cosmetics amp Toiletries 2004 119 49 58 Mas Chamberlin C Mondon P Lintner K Cosmetic management of lipid storage in adipocytes a slimming concept for men and women 2005 Cosmetic amp Toiletries Manufacture Worldwide 2005 53 56 Rotunda AM Avram MM Avram AS Cellulite Is there a role for injectables 2005 J Cosmet Laser Ther 7 3 4 147 154 onyder PB Emerging Therapeutic Targets 1999 3 4 587 599 onyder PB Esselstyn JM Loughney K Wolda SL Florio VA The role of cyclic nucleotide phosphodiesterases in the regulation of adipocyte lipolysis J Lipid Res 2005 46 494 503 van Vliet M Ortiz A Avram MM Yamauchi PS An assessment of traditional and novel therapies for cellulite 2005 J Cosmet Laser Ther 7 1 7 10 Rev July 2010 Page 11 of 112. 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 196 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 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 should be performed in triplicate 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 Incubate the plates at 37 C in a humidified incubator for 3 hours for time course experiments the longest time point is usually 24 hours One hour prior to the assay
3. requires screening many compounds and plant extracts Prior to beginning a clinical trial of the product one would need to establish validity of the lipolytic activity in human adipocytes Figure 1 Role of adipocytes in cellulite treatment nucleus Free fatty acid Free fatty acid F d ree fatty aci cellulite reduced cellulite appearance smaller fat cell Rev July 2010 Page 2 of 11 WHAT IS THE SCIENCE BEHIND THIS KIT 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 The sympathetic nervous system plays a key role in the regulation 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 one molecule of glycerol and 3 molecules of free fatty acids FFA increased lipolysis Phosphodiesterases PDE are enzymes that transform cAMP to 5 AMP 5 prime adenosine mo
4. 00 ul Wash Buffer to all wells OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 Add another 200 ul Wash Buffer Plate A Remove all media amp Wash Buffer Add 150 ul O00000000000 i R lI treatments controls to 3 wells at a time OPTION Add 100 969969000000 EID noe ana ne ul well compounds to a fresh plate without cells Oo OOO000000000 Add treatments 3 wells at a time Incubate 3 5 hours at 37 C One hour prior to assay reconstitute Glycerol Reagent A and prepare standards Keep all at room temp Plate A blank plate iti i 070700010 001070 0 0 Remove 100 ul well conditioned media CE eO SOC OOOO ECORS 000000000000 from Plate A to a blank assay plate sec cugoese E Add 100 ul glycerol standards to empty 000000000000 000000000000 wells Add 100 ul well reconstituted Glycerol Reagent A fefeeleJefejere e ee ERU to the plate including the glycerol standards at 500000000000 100ul well and optional plate without cells S An additional blank assay plate may be necessary for the assay of glycerol standards Incubate at 25 C room temperature for 15 minutes Pop the bubbles in each well l Measure the optical density of each well at 540 nm using a spectrophotometer plate reader Rev July 2010 Page 10 of 11 REFERENCES 1 Arner P J Endocrinol 1997 155 191 192 Greenway FL Bray GA Heber D Topical fat reduction Obes Res 1995 3 Suppl 4 561S 568S Hexsel D
5. e of subcutaneous adipose tissue is a major contributor to the appearance of cellulite The histological studies of subcutaneous tissues from men and women suggest that the fat lobules are larger and more vertical in women than men As a result these larger less restricted lobules can express outward against the dermis causing the bumps and dimples characteristic of cellulite The femoral subcutaneous fat deposits in women also tend to be more lipogenic and less lipolytic than abdominal subcutaneous or visceral fat due to the difference in the distribution of a and p adrenergic receptors on adipocytes in these different regions When these fat cells increase in size the skin compartment bulges which forms the noticeable dimpling or cottage cheese look These fat cells contain triglycerides which must be broken down before fat cells can be reduced in size The more triglyceride in fat is broken down the smaller the fat cells under the skin leaving the skin appearing smoother less cellulite Increased lipolysis or fat reduction of the subcutaneous adipose fat under the skin means more triglyceride is broken down to lead to smaller fat cells and a reduction of the cellulite appearance Topically applied lipolytic agents can distribute or reduce local fat accumulation and improve the aesthetic appearance of the skin Mas Chamberlin et al 2006 Hexsel et al 2005 Huber et al 2004 Testing lipolytic activity of potential treatments for cellulite
6. e oxidase to dihydroxyacetone phosphate DAP and hydrogen peroxide H2023 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 the glycerol concentration of the sample GLYCEROL ATP G 1 P ADP G 1 P O 5 DAP H O H20 4 AAP ESPA Quinoneimine dye H20 ITEMS INCLUDED IN THE KIT ITEM DESCRIPTION UNIT STORAGE Color Adipocytes Plate A Cultured human subcutaneous adipocytes o PaE 1 37 Blank Assay Plates 96 well assay plates blank poo PLATE 2 Assay Buffer 100 ml BormE 1 4 WashBufer om amp me 1 4 NN 0 196 DMSO in Assay Buffer GREEN 1 ml 20 C VIAL ul control Isoproterenol 10 mM in DMSO Dilute to 1 uM in Assay BLUE 10 ul 20 C Buffer before use i e 1 ul in 10 ml Assay Buffer VIAL BOTTLE m Glycerol Reagent Reconstitute with 11 0 ml deionized water prior to use MM For multi channel pipetters clear polyvinyl EAH 2 Glycerol standard Glycerol 1mM Reconstitute with 400 ul Wash Buffer ORANGE 100 ul 20 a to make the 200 uM glycerol standard see page 6 for VIAL recommended dilution scheme ALTERNATE 3 lsobutyl 1 methylxanthine IBMX 100 mM in DMSO 10 ul 20 C Positive contro
7. ge effects e 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 When do I need to use the IBMX positive control If 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 2 Can I 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 A REAGENTS ONLY kit is available cat LIP 1 NC Contact ZenBio to order additional cells or media 3 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 your order 4 have more samples plus standards to run than can fit on 1 96 well plate Can compare data obtained from multiple plates The lipolys
8. is 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 5 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 I 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 Rev July 2010 Page 8 of 11 APPENDIX A PLATE LAYOUT Rev July 2010 Page 9 of 11 APPENDIX B PROCEDURE FLOWCHART Remove 150ul 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 Buffer Plate A 000000000000 120 ul media 000000000000 OOOOO00000000 Remove 120 ul media from all wells Add 000000000000 OOOOO00000000 200 ul Wash Buffer to all wells 200 ul Wash Buffer Plate A 200 ul Wash Buffer Remove 200 ul media amp Wash Buffer Add another 2
9. l Dilute to 100 uM in Assay Buffer before use i e 1 ul in VIAL 1 ml Assay Buffer USE ONLY IF YOUR TREATMENT TIME EXCEEDS 5 HOURS 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 e Incubator at 37 C Large gauge needle e Option Step 5 of Assay Procedure 96 well plate blank Tubes for diluting glycerol standards Rev July 2010 Page 4 of 11 ASSAY PROCEDURE 1 Human 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 Plate A 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 are 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
10. n 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 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 July 2010 Page 7 of 11 TROUBLESHOOTING Problem Suggestions High background or the glycerol reagent A e Change pipet tips frequently turns purple before the assay begins e Use Glycerol Reagent A before the expiration date No response to positive control e Do not add the compounds and controls too fast The cells can float if a solution is added too fast e Make sure to starve the cells for 5 7 days BEFORE initiating treatment e DO NOT use IBMX as the positive control if you are incubating for less than 5 hours Ed
11. nophosphate 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 can be used as a positive control if your test compounds are suspected PDE inhibitors PDE inhibitors can be found as an ingredient in mesotherapy solution for the treatment of cellulite Snyder et al 2005 Isoproterenol a non specific B adrenergic agonist is used as the positive control if your test compounds affect lipolysis via B adrenergic receptors Among the methods for stimulating lipolysis the most commonly known and used is that which consists of inhibiting the phosphodiesterase in order to prevent or at least limit the rate of degradation of cyclic AMP In effect the phosphodiesterase destroys cyclic AMP by transforming it into 5 AMP so that it cannot function as a lipolysis activator Among the common agents for treatment of cellulite as slimming agents are xanthine analogs such as caffeine or theophylline These agents block the antilipolytic action of adenosine a potent endogenous inhibitor of lipolysis Other known methods in lipolysis stimulation are achieved by inhibiting phosphodiesterase in order to prevent or at least limit the degradation of cAMP Other existing methods for the treatment of cellulite have been the stimulation of adenylate cyclase to increase cAMP levels or to block the antilipolytic inactivation of adenylate cycla
12. 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 of wash buffer into 6 tubes not Rev July 2010 Page 5 of 11 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 pl 250 ul 250 ul 250 u1 250 pl S86 5686 200 100 12 5 6 25 3 125 uM x ed 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 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 otore 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 ligh
13. se a 2 adrenergic antagonists Greenway et al 1995 disclose that isoproterenol a known p agonist adrenergic stimulator is effective for the treatment of cellulite by stimulating lipolysis furthermore creams based on yohimbine a known a2 blocker applied to women s skin showed a decrease in thigh circumference Figure 2 Overview of adipocyte lipolysis EPINEPHRINE EPINEPHRINE NeR Bay Ba ABBREVIATIONS AC adenylate cyclase AR adrenergic receptors Ec G protein coupled receptor FFA free fatty acids PKA protein kinase AMP adenosine monophosphate ATP adenosine triphosphate 3 IR insulin receptor PKA EE PDE phosphodiesterase Per Perilipin dE 0 0 760 ewe 0000000 FFA glyc erol 7 WS FFA glycerol L5 bloodstream Rev July 2010 Page 3 of 11 WHAT DOES THIS KIT MEASURE This kit provides the tool to study chemical compounds that may influence lipolysis in cultured human adipocytes This kit specifically measures the free glycerol released by the breakdown of triglyceride The amount of free glycerol released from the cells is proportional to the ability of the test chemical to break down triglyceride 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 phosphat
14. t protected bottle Reconstituted Glycerol Reagent A is stable for 7 days 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 another blank plate This is most easily accomplished using a multi channel pipet Add 100 ul of each glycerol standard to any remaining empty wells in this plate or use the second blank plate provided in this kit for the standards 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 assay plates containing samples and standards 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 July 2010 Page 6 of 11 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 uM OD OD Sn Glycerol Standard Curve glycerol blank blank blank t temlam tf 0 043 6 25 0 020 25 0 205 0 164 200 oe98 0 697 0 656 0 655 0655 E c E e Series iie ar Series 100 150 200 250 uM Glycerol y observed O D minus the blank X concentratio
15. this product ORDERING INFORMATION AND T ECHNICAL SERVICES 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 1 866 ADIPOSE 866 234 7673 e Electronic mail e mail information zen bio com e World Wide W eb http www zenbio com Rev July 2010 Page 1 of 11 INTRODUCTION WHAT IS CELLULITE Cellulite is a term applied to a skin condition associated with the localized fat deposits that present as lumps and dimples appearing on the thighs of many women Although cellulite primarily afflicts the thighs hips and buttocks it may also be present on the stomach and upper arms Cellulite is simply made up of ordinary fatty tissue Fibrous strands called connective tissue which separate the skin from the underlying fatty tissue form separate compartments under the skin that contain fat cells The appearance is frequently described as orange peel skin or said to have a cottage cheese appearance Cellulite afflictions are a stubborn problem causing emotional and psychological distress to many women Although the etiology of cellulite is poorly understood the main factor appears to be local accumulation of fat in a regional compartment HOW CAN THIS KIT HELP MY RESEARCH Adipocytes fat cells are the principle cells implicated in fat storage by adipose tissue It has been proposed that the anatomical structur
16. zenbio gt Cellulite Treatment Screening Kit Human Adipocyte Lipolysis Assay Free Glycerol Detection Cat LIP 10 INSTRUCTION MANUAL 2ZBM0017 05 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 Glycerol Reagent A amp Buffers 4 C Use reconstituted Glycerol Reagent A within 7 days e Glycerol Standard amp Controls 20 C e Assay plate A 96 well cultured human adipocytes 37 C humidified incubator e Long term storage Remove the glycerol reagent A and buffers 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 express 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
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