(TG-5-RB) - Zen
Contents
1. 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 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 04 16 2014 Page 9 of 9 OO0000000000 OO00000000000 OO00000000000 OO00000000000 000000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 000000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 Standards 070 010 0 0101010 0100 070 010 0 010101010100 070 010 0 010101010100 070 010 0 010101010100 070 010 0 0101010 010 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 Buffer p 010 01010 010100010 0 010 010 0 010100010 0 010 010 0 010100 010 0 010 010 0 010100010 0 010 010 0 010 00 010 0 20 ul GLYCEROL REAGENT A An additional blank assay plate may be2. 6 25 0 07 0 07 0 022 0 022 0 070 0 500 12 5 0 098 0 098 0 05 0 05 0 098 25 0 127 0 13 0 079 0 082 0 129 0400 e Seriesi 50 0 2 0 205 0 152 0 157 0 203 OD blask Linear Series 1 100 0 353 0 362 0 305 0 314 0 358 0 300 200 0 649 0 667 0 601 0 619 0 658 0 200 0 100 slope 0 003 intercept 0 053 ia R 0 9997 0 50 100 150 200 250 Glycerol uM 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 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 04 16 2014 Page 5 of 9 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
3. 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 FREQUENTLY ASKED QUESTIONS 1 Can buy the reagents separately The only reagents sold separately are Glycerol Reagent A cat RGTA 10 RGTA 40 and the glycerol standard for the Triglyceride Assay kit cat TG GLYSTAN 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 Can 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 My cells did not lyse What can 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 do not have time to complete the assay Can I 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 Rev 04 16 2014 Page 6 of 9 PATEN
4. formats please adjust the volumes added to each well according to the surface area of the well flask you are using See your cultureware manufacturer s technical information for the specifications ITEMS INCLUDED IN THE KIT tem _____tnatruetions Amount OTY Storage WashBufer O O O OOOO CO se ooo f Reagent B Reconstitute w 10 ml deionized water 10ml 1 4 C Reconstitute w 2 5ml deionized water 2 5ml 1 4 C nal Combine and mix before use FOIRE Glycerol Glycerol 1mM Reconstitute with 400 ul 100 ul 3 20 C standard Standards Diluent to make the 200 uM glycerol standard see manual for recommended dilution scheme Standards 11 ml 1 4 C Other equipment reagents required but not provided with the kit Single channel pipetter Multi channel pipetter Plate reader with a filter of 540 nm Tubes to dilute glycerol standards Disposable reagent trays Mature adipocytes or other cells Clear bottom 96 well assay plates 1 1 Reagent A Reconstitute w 40 ml deionized water 40ml 1 Reconstitute w 11 ml deionized water 11ml 1 Combine and mix before use Use reconstituted Reagent within 7 days Rev 04 16 2014 Page 2 of 9 PATENT PROTECTED 6 153 432 ASSAY PROCEDURE Ts Warm the Wash buffer and Lysis buffer in a 37 C water bath Prepare the Reagent B by adding 10ml and 2 5 ml deionized water to the labeled bottles and gently invert DO NOT VORTEX Use a pipet to ensure that the powder is c
5. T PROTECTED 6 153 432 TROUBLESHOOTING Problem Suggestions High background or the triglyceride e Use clean tray and tips reagent turns a darker color before e Change pipet tips frequently the assay begins Edge effects e Ensure a saturated humidity in the incubator to prevent evaporation from the outside wells Inconsistent OD reading e 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 e Mix the lysates well before transferring the 20ul to the Wash buffer plate REFERENCES 1 Green H and Kehinde O 1974 Sublines of mouse 3T3 cells that accumulate lipid Cell 1 113 116 2 Hauner H et 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 04 16 2014 Page 7 of 9 PATENT PROTECTED 6 153 432 APPENDIX A PLATE LAYOUT Rev 04 16 2014 Page 8 of 9 PATENT PROTECTED 6 153 432 APPENDIX B 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 well Incubate 20 minutes at 37 50 C Verify lysis and add 135 ul warm Wash Buffer Mix by pipetting up and down 3 times Add 20 ul Reagent B and incubate 2 hours at 37 C
6. necessary for the assay of glycerol standards PATENT PROTECTED 6 153 432
7. ompletely dissolved Keep at room temperature Store in a light protected bottle Reconstituted Glycerol Reagent B is stable for 60 days refrigerated 2 8 C store any remaining solution refrigerated 2 8 C Remove all media from mature human adipocytes 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 disposable reagent tray not provided add 15 ul Lysis buffer per well Incubate at 37 C 50 C for 20 minutes 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 Sometimes pipet mixing is necessary for full lysis Add 135 ul warm Wash Buffer to each well 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 C for 2 hours One hour prior to the assay bring the glycerol standard and Reagent B to room temperature 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 depic
8. ted below Mix each new dilution thoroughly before proceeding to the next The 200 uM stock dilution serves as the highest standard the Diluent serves as the zero standard Rev 04 16 2014 Page 3 of 9 PATENT PROTECTED 6 153 432 400 pl 250 ul 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 this time prepare the Reagent A by adding 40 ml and 11 ml respectively of deionized water to the appropriate bottle and gently invert DO NOT VORTEX Use a pipet to ensure that the powder is completely dissolved Mix and combine the 2 volumes for use in the assay Keep at room temperature If using a Glycerol Reagent A solution previously prepared and stored at 2 8 C also bring to room temperature Make sure there is enough Glycerol 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 not provided 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 ver
9. y 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 13 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 microtiter plate reader Rev 04 16 2014 Page 4 of 9 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 OuM standard from all OD values including the standard curve Avg Standard Curve Glycerol OD OD OD uM OD OD blank blank _ blank 0 700 0 0 048 0 048 0 048 Cer 3 125 0 059 0 058 0 011 0 01 0 059 one
10. zenbio gt Triglyceride Assay Kit Bulk 500 Point Assay Kit 96 well format Cat TG 5 RB INSTRUCTION MANUAL ZBMO0014 05 STORAGE CONDITIONS e Reagents A amp B Buffers Store at 2 8 C Use reconstituted Glycerol Reagent A within 7 days 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 East NC 54 Suite 100 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 04 16 2014 Page 1 of 9 PATENT PROTECTED 6 153 432 The contents of this kit are sufficient for the assay of up to five 96 well plates 500 assay points The protocol is designed for assay of cells in a 96 well format For other
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