Wrinkle Treatment Screening Kit (DIF-10) - Zen
Contents
1. z L J 21 4 z L 4 sjueuoduio2 1siuoDe XINGI VANL 451 uvad XINGI OSING 10 1 eAnisod 1 SIN39V3d dO SNOILISOdWOO V XIaN3ddV Rev 8 18 2008 APPENDIX B PLATE LAYOUT 01 cl Rev 8 18 2008 Page 10 of 12 PATENT PROTECTED 6 153 432 APPENDIX C DIFFERENTIATION PICTURES PREADIPOCYTE MATURE ADIPOCYTE nucleus nucleus APPENDIX D DIFFERENTIATION FLOWCHART DAY 1 PLATE CELLS INCUBATE 24 HOURS 37 DAY 2 APPLY TREATMENTS IN INITIATION MEDIUM INCUBATE 7 DAYS 37 C DAY 8 FEED CELLS MAINTENANCE MEDIUM INCUBATE 7 DAYS 37 DAY 15 CELLS READY FOR ASSAY MOVE ON TO TRIGLYCERIDE ASSAY PROTOCOL Rev 8 18 2008 Page 11 of 12 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 Verify lysis and add 135 ul warm Wash Buffer Add 20 ul Reagent B and incubate 2 hours at 37 One hour prior to assay reconstitute Glycerol Reagent A and prepare standards Keep all at room temperature Add 80 Wash Buffer to a new plate Mix2. 10 Pipet 150 Positive Control PC 150 ul Negative Control NC and 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 100 ul media from all wells Gently feed all wells with 100 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 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 B Triglyceride Assay 1 Warm the Wash Buffer and Lysis Buffer in a 37 water bath Bring Reagent B to room temperature 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 room temperature Store in a light protected bottle Reconstituted Reagent B is stable for 60 days refrigerated 2 8 store any remaining solution refrigerated 2 8 C Bring Reagent B to room temperature 2 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 3 Remove all Wash Buffer
3. Using a new tray add 15 ul Lysis buffer Incubate at 37 50 for 20 minutes 4 After the incubation is complete visually confirm cell lysis by checking the wells under a microscope If cells are not fully lysed incubate for another 10 minutes 5 Add 135 ul warm Wash Buffer to each well 6 Add 20 ul Reagent B to each well It is not necessary to mix at this time however gently tap plate to help mix reagents Incubate the plate at 37 for 2 hours Rev 8 18 2008 Page 5 of 12 PATENT PROTECTED 6 153 432 7 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 Prepare the standard curve as follows Pipette 200 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 125 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 200 ul 1254 125 ul 125 ul 125 ul 1254 125 100 50 25 12 5 6 25 3 125 uM uM uM uM 8 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 pi
4. humidity The cells will be maintained in 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 supplied to prepare an intermediate concentration that would allow for a final volume of 10 ml Also the positive control in this kit the PPAR agonist has a final solvent concentration of 0 1 DMSO This 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 196 of any solvent as higher concentrations may be toxic to the cells Rev 8 18 2008 Page 4 of 12 PATENT PROTECTED 6 153 432 2 7 Twenty four hours later check the cells for confluence 8 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
5. 08 Page 1 of 12 PATENT PROTECTED 6 153 432 INTRODUCTION FAT AS YOUR FRIEND The increased demand for anti aging treatments has ranged from natural botanicals cosmetics to surgical intervention Majeed amp Prakash 2002 In the battle against the appearance of aging fat is the friend not the foe In addition to the treatment of facial wrinkles the transfer of one s own fat can be used to treat indented acne scars fill out the back of the hands and correct skin depressions Christensen et al 2005 WHY FAT okin is made up of 3 layers the epidermis the dermis and the subcutaneous layer The epidermis contains mainly keratinocytes which secrete keratin to provide the barrier function of skin The dermis contains sweat glands hair follicles and fibroblasts These fibroblasts are responsible for secreting collagen to provide elasticity for the skin The subcutaneous layer contains blood vessels nerve fibers and adipocytes These adipocytes provide the volume that helps add to the thickness and suppleness of skin associated with youth Loss of the fat cell layer that occurs with age or free radical damage results in the uneven depression in skin commonly observed as wrinkles and lines Considering the myriad of synthetic cosmetic filler products available one must ask why use fat Fat presents as an ideal soft tissue augmenter Adipose tissue is comprised of precursor cells called preadipocytes and adipocytes fat cells Stimulation of
6. 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 7096 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 PM 1 3 Centrifuge 1 200 rpm 282Xg 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 0 496 trypan blue solution We suggest withdrawing an aliquot of 50 of cells and mixing with 100 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 1504 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 incubator 5 97
7. d to show relative lipid accumulation of experimental treatments compared to controls The assay is based on the equation 1M 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 Subtract the OD value of the OuM standard from all OD values including the standard curve Zero blank 040 OD y 0 0034x 0 0075 F 0 9985 100 150 Glycerol in uM slope intercept 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 0 0075 0 003 where 0 003 slope of the line and 0 0075 y intercept Be careful to enter the proper sign for the y intercept value as it may be a negative number 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 oolve for the Total Glycerol concentration i e total triglyceride concentration for each OD Remember to include the Dilution Factor in the equation Data is express
8. ed 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 Also 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 more dilute solution of the condition medium at the time of the assay Rev 8 18 2008 Page 7 of 12 PATENT PROTECTED 6 153 432 TROUBLESHOOTING Problem ouggestions High background or the triglyceride reagent e Use clean tray and tips turns a darker color before the assay e Change pipet tips frequently begins Edge effects e Ensure a saturated humidity in the incubator to prevent evaporation from the outside wells Inconsistent OD reading Becareful when pipetting to avoid bubbles If bubbles persist burst the bubbles using a large gauge needle prior to reading and read the plate again Mix the lysates well before transferring the 20 to the Wash buffer plate Cells appear dead after 7 days treatment e acute treatment for days in Initiation Medium with my compound followed by additional feedings each 7 days should yield suitable positive control signal to complete the assay REFERENCES 1 Majeed M and Prakash L 2002 Novel natural approaches to anti aging skin care Cosmetics and Toiletries Manufacture Worldwide 2002 11 15 2 Christensen L Freiting V Janssen M Vuust J Hogdall E Adverse reactions to injectable s
9. 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 Reagent A to samples and standards Incubate 15 minutes at 25 room temperature Pop the bubbles in each well Measure the optical density of each well at 540 nm using a spectrophotometer plate reader Rev 8 18 2008 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOOO0000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOO0O00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 OOOOO00000000 Standards OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO Page 12 of 12 20 ul GLYCEROL REAGENT A All media Add 15 ul Lysis Buffer Add 135 ul warm Wash Buffer OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO OOOOOOOOOOOO 150 ul Wash Buffer 150 ul Wash Buffer Add 20 ul Reagent B Add 80 ul Wash Buffer An additional blank assay plate may be necessary for the assay of glycerol standards PATENT PROTECTED 6 153 432
10. oft tissue permanent fillers Aesthetic Plast Surg 2005 29 1 34 48 Rev 8 18 2008 Page 8 of 12 PATENT PROTECTED 6 153 432 XIN8I ululuexI ul uul nqosi uioAulo1daJ1S uio Aulo1daJ1S PATENT PROTECTED 6 153 432 Page 9 of 12 5 oeuoseujeulexe ul INSUI ullog uloig 619 9UIAOQ 619 191 SdddH SdddH SdddH z L 4 5 WANG z L 4 5 NING z 4 NING sjueuoduio INI NIN s u e y D JN L jsiuoDe AYWdd jsiuoDe AYWdd OSING XIN8I 1 3 XIN8I 1 3 3 uiouejouduuy lt ouoseujeulexe euoseujeulexe euoseujeujexe UBLUNH unoig umu s 619 J 2194 e1eJ SdddH SdddH SdddH
11. pet 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 is stable for 60 days refrigerated 2 8 C store any remaining solution refrigerated 2 8 C 9 Toa 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 10 Working with one row or column at a time mix the lysates very well using a multi channel pipet Immediately transfer 20 per well of the lysates to the corresponding well of the plate containing the wash buffer This results in a Dilution Factor of 5 11 Prepare the standard curve Pipet 100 ul of each standard into a well NOTE Eight wells are necessary for the curve there are remaining wells on the assay plate you can utilize the remaining wells If not a second plate is included in this kit 12 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 13 Read at 540 nm using a microtiter plate reader Rev 8 18 2008 Page 6 of 12 PATENT PROTECTED 6 153 432 GLYCEROL STANDARD CURVE This kit is designe
12. ssay is total triglyceride content of the cells 2 weeks after the initiation of differentiation ITEMS INCLUDED IN THE KIT Item Description Unit Quantity Item Storage Human SQ Human subcutaneous preadipocytes gt 2 0 X10 VIAL 1 1 Liquid som 2 46 sm 2 4 ec Glycerol standard Glycerol 1mM Reconstitute with 200 ul VIAL 50 ul 2 20 C Standards Diluent to make the 200 uM glycerol standard see page 5 for recommended dilution scheme 2 zm 4 Clear polyvinyl tray for muli channel pipetters exw 3 Data shest Certificate of Analysis and protocol _______ 96 well plate Tor plating and diferertiaing eme 1 Assay Plate Plats 96 well plate blank for samples amp standards se 2 _ 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 w w w w Rev 8 18 2008 Page 3 of 12 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
13. the preadipocytes in the subcutaneous skin layer to differentiate into fat cells is called adipogenesis By studying products capable of triggering or enhancing the differentiation of preadipocytes into fat cells one can produce a cosmetic product designed to work with a person s own body to naturally fill in deficient areas of the skin For example the creation of fat cells in an aged face can provide a natural augmenter to fill out the fine lines and wrinkles of the face Skin would have increased thickness and volume thereby resulting in smoother younger looking skin BEFORE AFTER wrinkled skin smooth skin A B C PREADIPOCYTES FAT CELLS FAT CELLS A Epidermis skin surface B Dermis C Subcutaneous layer Rev 8 18 2008 Page 2 of 12 PATENT PROTECTED 6 153 432 WHAT DOES THIS MEASURE The differentiation assay kit provides 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 stimulation and maintenance of human fat cells This kit is designed to test compounds as potential 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 The end point a
14. zenbio Wrinkle Treatment Screening Kit Human Adipocyte Differentiation Assay Cat DIF 10 INSTRUCTION MANUAL 2ZBM0020 01 STORAGE CONDITIONS e Frozen human preadipocytes otore 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 e Media Reagents amp B Buffers otore at 2 8 See kit label for expiration date NOTE expiration is 4 weeks from date of manufacture e Glycerol Standard 20 C For Research Use Only Not For Use In Diagnostic 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 Zen Bio Inc 3200 Chapel Hill Nelson Blvd Suite 104 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 8 18 20
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