Glucocorticoid Analogues (DIF-GLUC) - Zen
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1. Assay Plate blank 96 well assay plates blank PLATE 3 Other equipment reagents required but not provided with the kit e Single channel pipetter Multi channel pipetter e Plate reader with a filter of 540 nm Tubes to dilute standards Glycerol standard Rev July 2010 Page 2 of 15 PATENT PROTECTED 6 153 432 ASSAY PROCEDURE We strongly recommend testing all compounds in triplicate 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 Any deviation from the recommended start day of Monday Thursday may require weekend work 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 Upon thawing transfer the cells to a sterile conical bottom centrifuge tube containing 10 ml of Preadipocyte Medium cat PM 1 Centrifuge 1 200 rpm 282Xg 20 C 5 minutes Aspirate the supernatant TAKE CARE TO NOT ASPIRATE ANY OF THE CELL PELLET The cell vial contains a minimum of 2 0 x 10 viable cells however we recommend performing a cell count to determine a more e2. 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 can 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 5 I1 do 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 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 prevent Edge effects evaporation from the outside wells Be careful when pipetting to avoid bubbles If bubbles persist burst the bubbles using a large gauge needle prior Inconsistent OD reading to reading and read the plate again Mix the lysates well before transferring the 10ul to the Wash buffer plate Rev July 2010 Page 9 of 15 PATENT PROTECTED 6 153 432 REFERENCES 1 Green H and Kehinde O 1974 Sublines of mouse 3T3 cells that accumulate lipid Cel 1 113 116 2 Hauner H et
3. al 1989 J Clin Invest 84 1663 1670 3 Kuri Harcuch W Wise LS Green H 1978 Interruption of the adipose conversion of 3T3 cells by biotin deficiency differentiation without triglyceride accumulation Cell 14 53 58 Rev July 2010 Page 10 of 15 PATENT PROTECTED 6 153 432 APPENDIX A COMPOSITION OF REAGENTS Reagent Preadipocyte Medium Differentiation Positive Control DPC agonist dexamethasone Differentiation Negative Control DNC agonist dexamethasone TNF a Differentiation Vehicle Control DVC agonist no dexamethasone Differentiation Medium for Treatment compound DMT agonist compound Rev July 2010 Components Page 11 of 15 DMEM Ham s F 12 medium HEPES Fetal bovine serum Penicillin Streptomycin Amphotericin B DMEM Ham s F 12 medium HEPES Fetal bovine serum Biotin Pantothenate Human insulin Penicillin Streptomycin Amphotericin B Isobutylmethylxanthine IBMX PPARY agonist Dexamethasone DMEM Ham s F 12 medium HEPES Fetal bovine serum Biotin Pantothenate Human insulin Penicillin Streptomycin Amphotericin B Isobutylmethylxanthine IBMX PPARY agonist Dexamethasone TNF a DMEM Ham s F 12 medium HEPES Fetal bovine serum Biotin Pantothenate Human insulin Penicillin Streptomycin Amphotericin B Isobutylmethylxanthine IBMX PPARY agonist DMEM Ham s F 12 medium HEPES Fetal bovine serum Biotin PATENT PROTECTED
4. this time prepare the Reagent A by adding 11 0 ml deionized water per bottle and gently inverting 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 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 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 pipet 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 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 ul 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 microti
5. 0000 O00000000000 O00000000000 O00000000000 000000000000 Standards 00 0 0 0 000 0 09 0 00 0 0 0 000 0 09 0 00 0 0 0 000 0 09 0 00 0 0 0 0000 009 0 00 0 0 0 0 00 0 009 0 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 ul Wash ac Buffer 00 0 0 0 0 00 0 09 0 00 0 0 0 00 0 0 009 0 00 0 0 0 000 0 009 0 000000000000 20 ul 000000000000 GLYCEROL REAGENT A An additional blank assay plate may be necessary for the assay of glycerol standards PATENT PROTECTED 6 153 432
6. 6 153 432 Differentiation Medium for Treatment compound DMT agonist compound continued Maintenance Medium Positive Control MMPC dexamethasone Maintenance Medium Negative control MMNC dexamethasone TNF a Maintenance Medium Vehicle control MMVC no dexamethasone Maintenance Medium Treatment compounds MMT compound Rev July 2010 Pantothenate Insulin Penicillin Streptomycin Amphotericin B lsobutylmethylxanthine IBMX PPARY agonist Compound DMEM Ham s F 12 medium HEPES Fetal bovine serum Biotin Pantothenate Insulin Penicillin Streptomycin Amphotericin B Dexamethasone DMEM Ham s F 12 medium HEPES Fetal bovine serum Biotin Pantothenate Insulin Penicillin Streptomycin Amphotericin B Dexamethasone TNF a DMEM Ham s F 12 medium HEPES Fetal bovine serum Biotin Pantothenate Insulin Penicillin Streptomycin Amphotericin B DMEM Ham s F 12 medium HEPES Fetal bovine serum Biotin Pantothenate Insulin Penicillin Streptomycin Amphotericin B Compound Page 12 of 15 PATENT PROTECTED 6 153 432 APPENDIX B PLATE LAYOUT Rev July 2010 Page 13 of 15 PATENT PROTECTED 6 153 432 APPENDIX C DIFFERENTIATION PICTURES PREADIPOCYTE MATURE ADIPOCYTE nucleus APPENDIX D DIFFERENTIATION FLOWCHART Day 1 PLATE CELLS INCUBATE 24 HOURS 37 C l DAY 2 APPLY TREATMENTS AND CONTROLS INCUBATE 7 DAYS 37 C DAY 8 CHANGE
7. CELLS TO APPROPRIATE MAINTENANCE MEDIUM INCUBATE 7 DAYS 37 C l Day 15 FEED CELLS APPROPRIATE MAINTENANCE MEDIUM l MOVE ON TO TRIGLYCERIDE ASSAY PROTOCOL Rev July 2010 Page 14 of 15 PATENT PROTECTED 6 153 432 APPENDIX E TRIGLYCERIDE ASSAY FLOWCHART__ Remove all media from wells l Wash with 150 ul Wash Buffer Remove all Wash Buffer from wells and add 15 ul Lysis Buffer i Incubate 20 minutes at 37 50 C Verify lysis and add 135 ul warm Wash Buffer Mix by pipetting up and down 3 times l Add 20 ul Reagent B and incubate 2 hours at 37 C i 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 Mix lysates and transfer 20 ul lysates to the wells containing Wash Buffer Transfer 100 ul of each standard to a new plate l Add 100 ul Reagent A to samples and standards l Incubate 15 minutes at 25 C room temperature Pop the bubbles in each well5 l Measure the optical density of each well at 540 nm using a spectrophotometer plate reader Rev July 2010 Page 15 of 15 O00000000000 O00000000000 O00000000000 O00000000000 000000000000 O00000000000 O00000000000 O00000000000 O00000000000 000000000000 O00000000000 O00000000000 O00000000000 O00000000000 000000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 O0000000
8. concentration that would allow for a final volume of 10 ml Also the positive control in this kit dexamethasone has a final solvent concentration of 0 00596 ethanol This low concentration does not affect the differentiation of adipocytes so the ethanol is not included in the vehicle control If the concentration of any solvent for the compounds used is high enough to potentially alter differentiation please include the solvent alone as a treatment We do not recommend treating the cells with solutions exceeding 196 of any solvent as higher concentrations may be toxic to the cells Day 2 Begin the differentiation procedure using the 4 types of Differentiation reagents see summary chart above and Appendix A Plan to do all treatments and controls in triplicate A blank plate map is included in these instructions to record the well treatments Using the Differentiation Medium for Treatment DMT prepare treatments Refer to the note above when preparing compound solutions 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 Pipet 150 ul each Differentiation Positive Control DPC Differentiation Negative Control DNC and Differentiation Vehicle Control DVC into appropriate wells Remove media from experimental wells and pipet 150 ul each Differentiation Medium for Treatment DMT containing compounds into appropriate wells Rev J
9. il e mail information zen bio com e World Wide Web http www zenbio com Rev July 2010 Page 1 of 15 PATENT PROTECTED 6 153 432 INTRODUCTION The differentiation assay kits provide the tools to study the compounds that stimulate 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 glucocorticoid analogues with dexamethasone at 1 0 uM serving as the positive control This kit contains sufficient reagents to assay 100 assay points in a 96 well format ITEMS INCLUDED IN THE KIT Item Description Unit Quantity Item Storage Human Preadipocytes Human subcutaneous preadipocytes VIAL 1 Frozen vial Preadipocyte medium See Appendix A Differentiation Positive Control Differentiation Negative Control Liquid nitrogen Maintenance Medium for Negative Control MMVC Wash buffer Bore 50m 2 4C Lysis buffer BoE 15m 2 4C Reconstitute w 11 0 ml deionized water Reagent A prior to use BOTTLE 11ml 2 4 C Reagent B He uS w 2 5 ml deionized water prior Glycerol 1mM Reconstitute with 400 ul Standards Diluent to make the 200 uM glycerol standard see page 5 for recommended dilution scheme Standards Diluent BoE 2m 4C Data sheet Certificate of Analysis and protocol EacH 1
10. l 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 slope 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 8 of 15 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 FREQUENTLY ASKED QUESTIONS 1 Can 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 I 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 3 Can I use this kit to measure total triglyceride in other cell lines and other
11. lly confirm cell lysis by checking the wells under a microscope If cells are not fully lysed incubate for another 10 minutes Add 135 ul warm Wash Buffer and mix the lysates by pipetting up and down three times 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 2 hours 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 Rev July 2010 Page 6 of 15 PATENT PROTECTED 6 153 432 400 ul 250l 250 pl 250 ul 250 ul 250 ul 250 ul 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 9 Also at
12. ter plate reader Rev July 2010 Page 7 of 15 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 equation 1 M 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 O 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 as 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 i intercept 0 053 5008 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 glycero
13. uly 2010 Page 4 of 15 PATENT PROTECTED 6 153 432 Day 8 Prepare treatments using the Maintenance Medium Treatment Compounds MMT Using a multi channel pipetter remove media from all wells Gently feed all wells with 150ul of the appropriate Maintenance Medium see chart above that is provided with this kit Day 15 Cells are now mature Proceed to part B The positive control wells should exhibit significantly greater lipid accumulation than the negative control wells or the vehicle control wells Refer to page 11 for a picture of a typical positive control when the adipocytes are mature Rev July 2010 Page 5 of 15 PATENT PROTECTED 6 153 432 B TRIGLYCERIDE ASSAY 1 Warm the Wash Buffer and Lysis buffer in a 37 C water bath Prepare the Reagent B by adding 2 5ml deionized water per bottle and gently invert DO NOT VORTEX Use a pipette to ensure that the powder is completely dissolved Keep at 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 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 Remove all Wash Buffer Using a new tray add 15 ul Lysis buffer Incubate at 37 C 50 C for 20 minutes After the incubation is complete visua
14. xact number of cells Resuspend the cell pellet in 2 ml Preadipocyte Medium dilute an aliquot in trypan blue and count live unstained cells on a hemacytometer The cell concentration required for approximately 40 000 cells cm in the 96 well format with 150ul well is 1 3 x 10 cells in 15 ml Preadipocyte Medium Plate cells in one of the 96 well plates provided in the kit Do not agitate the plate as cells will not plate evenly Place plate in 37 C incubator 5 CO 97 humidity The cells will be maintained in the incubator after each manipulation until Day 14 Rev July 2010 Page 3 of 15 PATENT PROTECTED 6 153 432 GUIDE TO REAGENTS Well type of wells Differentiation reagent Maintenance medium Positive control 3 DPC MMPC Negative control 3 DNC MMNC Vehicle control 3 DVC MMVC Treatment compounds 87 DMT MMT Include any necessary solvent controls as treatments see Note below See Appendix A for description of reagents NOTE Included in this kit are sufficient volumes of Differentiation Medium for Treatments DMT and Maintenance Medium for Treatments MMT based on using 10 ml of each medium per compound dilution for a maximum of 29 compounds tested in triplicate 87 wells remaining on a 96 well plate after accounting for 9 control wells If a compound stock is too concentrated to accomplish the desired dilution use an appropriate solution not supplied to prepare an intermediate
15. zenbio gt Human Adipogenesis Assay Kit Glucocorticoid Analogues 100 point assay kit Cat DIF GLUC DIF GLUC NC INSTRUCTION MANUAL ZBM0004 05 STORAGE CONDITIONS Frozen subcutaneous human 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 See kit label for expiration date Use reconstituted Glycerol Reagent A 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 ma
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