Human Adipogenesis Assay Kit PPAR γ Agonists - Zen
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1. Human Adipogenesis Assay Kit PPAR y Agonists Cat DIF AG DIF AG NC INSTRUCTION MANUAL ZBMO0005 05 STORAGE CONDITIONS Frozen subcutaneous preadipocytes Store in liquid nitrogen IMMEDIATELY upon receipt No expiration date is applicable however the cells must be plated within 1 week of receiving the kit to account for the expiration of the kit components Media Reagents A amp B Buffers Store at 2 8 C Glycerol Standard 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 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 13 PATENT PROTECTED2. 6 153 432 INTRODUCTION The differentiation assay kits provide the tools to study the compounds that stimulate cultured human adipocyte differentiation or lipogenesis Such compounds may be PPARy agonists or a combination of thiazolidinediones and glucocorticoids that are potentially useful in the treatment of diabetes This kit is designed to test compounds as potential PPARy agonists It is our experience that the PPARy agonist used as the Positive Control sufficiently stimulates human adipocyte differentiation after 7 days of treatment The protocol is designed so that the test compounds are also used in a 7 day treatment regimen This kit contains sufficient reagents to assay 100 assay points in a 96 well format ITEMS INCLUDED IN THE KIT Item Description Unit Item Storage Human SQ Human subcutaneous preadipocytes gt 2 0 Liquid 6 Preadipocytes X10 cells vial cryopreserved Preadipocyte medium See Appendix sorter Negative control Positive control 2 49 Initiation medium See Appendix A 300m 3 46 MM Maintenance medium See Appendix A Bome 100m 2 4 2 4 2 4 Reconstitute w 11 0 ml deionized water prior rane Reagent savas use 11 0ml Reagent B w 2 5 ml deionized water prior Glycerol 1mM Reconstitute with 400 ul Glycerol standard Standards Diluent to make the 200 uM Via 100 wl 20 C glycerol
3. OSa 3 e e 9 1 4 z L 4 5 WANG OSING 1 0 XINGI OA 10 s usuodwo4y SLNJ9VJ4 40 SNOILISOdINOD XIGNAddV 153 432 PATENT PROTECTED 6 Page 10 of 13 Rev July 2010 APPENDIX C PLATE LAYOUT Rev July 2010 Page 11 of 13 PATENT PROTECTED 6 153 432 APPENDIX D DIFFERENTIATION FLOWCHART DAY 1 PLATE CELLS INCUBATE 24 HOURS 37 C l DAY 2 APPLY TREATMENTS IN INITIATION MEDIUM INCUBATE 7 DAYS 37 C DAY 8 CHANGE CELLS TO MAINTENANCE MEDIUM INCUBATE 7 DAYS 37 C DAY 15 CELLS ARE MATURE MOVE ON TO TRIGLYCERIDE ASSAY PROTOCOL Rev July 2010 Page 12 of 13 PATENT PROTECTED 6 153 432 APPENDIX E TRIGLYCERIDE ASSAY FLOWCHART__ Remove all media from wells Wash with 150 ul Wash Buffer Remove all Wash Buffer from wells and add 15 ul Lysis Buffer Incubate 20 minutes at 37 50 C Verify lysis add 135 ul warm Wash Buffer Mix by pipetting up and down 3 times Add 20 ul Reagent incubate 2 hours at 37 C One hour prior to assay reconstitute Glycerol Reagent A and prepare standards Keep all at room temperature Add 80 ul Wash Buffer to a new plate
4. allow for a final volume of 10 ml Also the positive control in this kit the PPARy agonist has a final solvent concentration of 0 1 DMSO This concentration is included in the vehicle control VC If the concentration of any solvent for the compounds used is high enough to potentially alter differentiation please include that solvent concentration as an additional treatment We do not recommend treating the cells with solutions exceeding 1 of any solvent as higher concentrations may be toxic to the cells Day 2 Twenty four hours later check the cells for confluence Using the Initiation Medium IM prepare treatments Plan to do all treatments in triplicate A blank plate map is included in these instructions to record the well treatments 9 When all treatments are prepared remove Preadipocyte medium from control wells We recommend doing the treatments in small groups so the cells do not dry out 10 Pipet 150 ul Positive Control PC 150 ul Negative Control NC 150 ul Vehicle Control VC into appropriate wells 11 Remove media from experimental wells and pipet 150 ul each treatment media into appropriate wells Incubate the plate for 7 days Day 8 12 Using a multichannel pipetter remove 90 ul media from all wells Gently feed all wells with 120 ul of the Maintenance Medium MM that is provided with this kit Incubate the plate for 7 days Day 15 13 Cells are now mature Proceed to part B 14 The positive c
5. lysates and transfer 20 ul lysates to the wells containing Wash Buffer Transfer 100 ul of each standard to a new plate Add 100 ul Glycerol Reagent A to samples and standards Incubate 15 minutes at 25 C room temperature Pop the bubbles in each well Measure the optical density of each well at 540 nm using a spectrophotometer plate reader Rev July 2010 Page 13 of 13 _ Standards 0000000000000 0000000000000 0000000000000 0000000090000 0009000000000 20 GLYCEROL REAGENT A All media 150 ul Wash Buffer 150 ul Wash Buffer Add 15 ul Lysis Buffer Add 135 ul warm Wash Buffer Add 20 ul Reagent B Add 80 Wash Buffer addition
6. standard see page 5 for recommended dilution ommers amon seeme O0 0 Bome am 4 _ Clear polyvinyl tray for muli channelpipetters 8 Data sheet Certfcate of Analysis and protocol en 1 Assay Plates blank 96 well assay plate blank eme 3 Other equipment reagents required but not provided with the kit Additional Maintenance Medium if necessary see background information Single channel pipetter Multi channel pipetter Plate reader with a filter of 540 nm Tubes to dilute glycerol standards v v v v Rev July 2010 Page 2 of 13 PATENT PROTECTED 6 153 432 ASSAY PROCEDURE A DIFFERENTIATION PROCEDURE On each day of the procedure the appropriate medium must be warmed to 37 C prior to use Note This protocol is designed to accommodate a weekday work schedule if started on a Monday Thursday Any deviation may require weekend work We strongly recommend testing all compounds in triplicate Day 1 This is the day the cells are plated 1 Remove cells from liquid nitrogen and place immediately into a 37 C water bath and agitate while in bath Be careful not to submerge the cap of the vial into water Do not leave the vials in water bath after most of the content has thawed Rinse the vials with 70 ethanol before taking them to the culture hood 2 Upon thawing transfer the cells to a sterile conical bottom centrifuge tube containing 10 ml of Preadipocyte medium cat
7. 2 hours 8 Bring Reagent A and the glycerol standards to room temperature during this time The Wash Buffer can also be kept at room temperature at this point Warm the Standards Diluent to 37 C Prepare the standard curve as follows Pipette 400 ul of the Standards Diluent 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 diluent 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 diluent serves as the zero standard 400 pl 250 ul 250 pl 250 pl 250 250 pl 250 pl Standards Diluent 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 Rev July 2010 Page 5 of 13 PATENT PROTECTED 6 153 432 9 Also at this time prepare the Reagent A by adding 11 0 ml deionized water per bottle and gently invert DO NOT VORTEX Use a pipet to ensure that the powder is completely dissolved Keep at room temperature If using a Reagent A solution previously prepared and stored at 2 8 C also bring to room te
8. ADIPOCYTE nucleus Rev July 2010 Page 9 of 13 PATENT PROTECTED 6 153 432 Sulyjuex AUJaw Ajngos e ullua d ul NSUI 1 913019 9 2 5 SWEH NIWA winipay 151 3 19 01494 SUOSEUJSWeX9q ul NSUI SUIAO 219 NANG 151 XG Od 103 09 e o 5 e eyeusyo Uue e WNJOS S3d3H z L 4 WANG 1 AYWdd e o ulju d uoseyz wex q ullolg WNJOS 2194 21 5 WING 4 1 451 ON 1 g ulnuejoudwy e SUIAO 2 NANG
9. PM 1 Centrifuge 1 200 rom 282 9 20 5 minutes Aspirate the supernatant TAKE CARE TO NOT ASPIRATE ANY OF THE CELL PELLET 4 The cell vial contains a minimum of 2 0 x 10 viable cells however we recommend performing a cell count to determine a more exact number of cells Resuspend the cell pellet in 2 ml Preadipocyte medium dilute an aliquot in 0 4 trypan blue solution We suggest withdrawing an aliquot of 50 ul of cells and mixing with 100 ul of the trypan blue solution resulting in a dilution factor of 3 Count live unstained cells on a hemacytometer The cell concentration required for approximately 40 000 cells in the 96 well format with 150uI well is 1 3 x 10 cells in 15 ml Preadipocyte medium 5 Plate cells in one of the 96 well plates provided in the kit Do not agitate the plate as cells will not plate evenly 6 Place plate in 37 C incubator 5 CO2 97 humidity The cells will be maintained the incubator after each manipulation until Day 14 NOTE This kit contains a sufficient volume of Initiation medium IM to use 10 ml of medium per compound dilution for a maximum of 29 compounds tested in triplicate 87 wells on the 96 well plate leaving 9 wells for controls If a compound stock is too concentrated to accomplish the desired dilution use an appropriate solution not Rev July 2010 Page 3 of 13 PATENT PROTECTED 6 153 432 supplied to prepare an intermediate concentration that would
10. al blank assay plate may be necessary for the assay of glycerol standards PATENT PROTECTED 6 153 432
11. e of the line and 0 0006 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 Rev July 2010 Page 7 of 13 PATENT PROTECTED 6 153 432 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 Solve for the Total Glycerol concentration i e total triglyceride concentration for each OD Remember to include the Dilution Factor in the equation Data is expressed as uM Glycerol NOTE Any OD values that are negative after the blank is subtracted should be considered to be 0 for the OD value TROUBLESHOOTING Problem Suggestions High background or the triglyceride Use clean tray and tips reagent turns a darker color before Change pipet tips frequently the assay begins Ensure a saturated humidity in the incubator to Edge effects prevent evaporation from the outside wells Be careful when pipetting to avoid bubbles If bubbles persist burst the bubbles using a large gauge needle prior to reading and read the plate again Inconsistent OD reading An acute treatment for 3 days in Initiation Medium Cells appear dead after 7 days followed by additiona
12. l feedings each 7 days should treatment with my compound yield suitable positive control signal to complete the assay FREQUENTLY ASKED QUESTIONS 1 I buy the reagents separately The only reagents sold separately are Glycerol Reagent A cat RGTA 10 and the glycerol standard for the Triglyceride Assay kit cat TG GLYSTAN 2 Can use another plate format besides 96 well This kit is designed for the assay of A 96 well plate 100 assay points We do not have a protocol for other formats I use this kit to measure total triglyceride in other cell lines and other human and non human cells Yes The assay is not species specific As long as the sample concentration is in the linear range this kit should be able to detect it 4 My cells did not lyse What I do If cells are not fully lysed incubate for another 10 minutes at 37 C 50 C Sometimes mixing by pipetting up and down several times is necessary for full lysis Rev July 2010 Page 8 of 13 PATENT PROTECTED 6 153 432 5 1 not have time to complete the assay Can freeze the samples Yes The cell lysates can be stored at 80 C for a maximum of 7 days Mix the thawed lysates in the plate by pipetting up and down several times Allow all reagents and samples to reach room temperature BEFORE adding the Wash Buffer and Glycerol Reagent A to complete the assay APPENDIX A DIFFERENTIATION PICTURES PREADIPOCYTE _ gt MATURE
13. mperature 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 10 To a blank 96 well plate add 80 ul wash buffer to each well needed for the assay NOTE do not add Wash Buffer to the wells used for the standard curve 11 Working with one row or column at a time mix the lysates very well using a multi channel pipette Immediately transfer 20 ul per well of the lysates to the corresponding well of the plate containing the wash buffer This results in a Dilution Factor of 5 12 Prepare the standard curve Pipet 100 ul of each standard into a well NOTE Eight wells are necessary for the curve or sixteen if performing the curve in duplicate If there are remaining wells on the assay plate you can utilize the remaining wells If not a second plate is included in this kit 13 Using the third tray add 100 Reagent A to samples and standards Mix by pipetting up and down one time Incubate at room temperature for 15 minutes 14 Read at 540 nm using a microtiter plate reader Rev July 2010 Page 6 of 13 PATENT PROTECTED 6 153 432 GLYCEROL STANDARD CURVE This kit is designed to show relative lipid accumulation of experimental treatments compared to controls The assay is based on the equati
14. on 1 Triglyceride yields 1M glycerol Free Fatty Acids The reagent measures the concentration of glycerol released after lysing the cells and hydrolyzing the triglyceride molecules The triglyceride concentration can then be determined from the glycerol values Generate standard curve see example below DO NOT use this standard curve to generate your data This is an example e Subtract the OD value of the 0 uM standard from all OD values including the standard curve Avg Glycerol OD OD OD uM OD OD blank blank blank 0 700 0 0 048 0 048 0 048 3 125 0 059 0 058 0 011 0 01 0 059 0 600 6 25 0 07 0 07 0 022 0 022 0 070 12 5 0 098 0 098 0 05 0 05 0 098 25 0 127 0 13 0 079 0 082 0 129 50 0 2 0 205 0 152 0 157 0 203 100 0 353 0 362 0 305 0 314 0 358 P 200 0 649 0 667 0 601 0 619 0 658 0 200 slope 0 003 intercept 0 053 R 0 9997 Standard Curve 100 150 200 Glycerol uM 250 y 0 003x 0 053 R 0 9997 Series Linear Series 1 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 0006 0 0014 where 0 0014 slop
15. ontrol wells should exhibit significantly greater lipid accumulation than the negative control wells or the vehicle control wells Refer to page 9 for a picture of a typical positive control when the adipocytes are mature TRIGLYCERIDE ASSAY 1 Warm the Wash buffer and Lysis buffer in a 37 C water bath 2 Prepare the Reagent B by adding 2 5ml deionized water per bottle and gently invert DO NOT VORTEX Use a pipet to ensure that the powder is completely dissolved Keep at Rev July 2010 Page 4 of 13 PATENT PROTECTED 6 153 432 room temperature Store in a light protected bottle Reconstituted Reagent B is stable for 60 days refrigerated 2 8 C store any remaining solution refrigerated 2 8 C Bring Reagent B to room temperature 3 Remove all media Using about 15 ml of the wash buffer wash the cells one time with 150 ul wash buffer Label the disposable tray wash buffer and retain for later use 4 Remove all Wash buffer Using a new tray add 15 ul Lysis buffer Incubate at 37 C 50 C for 20 minutes 5 After the incubation is complete visually confirm cell lysis by checking the wells under a microscope If cells are not fully lysed incubate another 10 minutes 6 Add 135 ul warm Wash Buffer and mix the lysates by pipetting up and down three times 7 Add 20 ul Reagent B to each well It is not necessary to mix at this time however gently tap the plate to help mix the reagents Incubate the plate at 37 C for
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