(TG-1-NC) - Zen
Contents
1. 0 619 0 658 slope 0 003 intercept 0 053 R 0 9997 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 OD blask 0 700 0 600 0 500 0 400 0 300 0 200 0 100 0 000 Standard Curve 100 150 200 Glycerol uM 250 y 0 003x 0 053 R 0 9997 Series1 Linear Series 1 Be careful to enter the proper sign for the y intercept value as it may be a negative number Rev 04 16 14 Page 5 of 9 PATENT PROTECTED 6 153 432 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 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 02. 400 pl 250 ul 250 pl 250 pl 250 pl 250 wl 250 pl 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 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 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 or sixteen if performing the curve in dupl
3. Reagent B to room temperature 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 new tray add 15 ul Lysis buffer to each 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 another 10 minutes 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 the 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 A 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 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 04 16 14 Page 3 of 9 PATENT PROTECTED 6 153 432
4. ffer Transfer 100 ul of each standard to a new plate Add 100 ul 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 14 OO00000000000 OO0000000000 OO00000000000 OO0000000000 OO00000000000 OO0000000000 OO0000000000 OO0000000000 OO0000000000 OO0000000000 OO0000000000 OO0000000000 OOO0000000000 OO0000000000 OO0000000000 OO00000000000 OO00000000000 OO00000000000 OO00000000000 000000000000 O00000000000 O00000000000 O00000000000 O00000000000 O00000000000 Standards 070 010 01010101010100 00 0 0 0 010101010100 00 010 01010101010100 070 010 0 0101010101010 00 010 0 0101010 0100 Page 9 of 9 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 ee Buffer 010 01010 010100 010 0 010 01010 010100 010 0 010 01010 010100010 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
5. for the OD value 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 14 Page 6 of 9 PATENT 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 FREQUENTLY ASKED QUESTIONS 1 Can 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 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 othe
6. icate 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 microtiter plate reader Rev 04 16 14 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 Glycerol OD OD OD uM OD OD blank blank blank 0O 0 048 0 048 0 048 3 125 0 059 0 058 0 011 0 01 0 059 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 0 358 200 0 649 0 667 0 601
7. itional reagents or formats please order our bulk 500 point assay kit cat TG 5RB ITEMS INCLUDED IN THE KIT ITEM DESCRIPTION UNIT QTY STORAGE Wash bufer BOTTLE SOM aeo yssir om TSM Glycerol Reagent Reconstitute w 11 0 ml deionized water BOTTLE 11ml x A prior to use Store at 4 C Use reconstituted Reagent B Reconstitute w 2 5 ml deionized water 2 eae ml prior to use TG Glycerol Glycerol 1mM Reconstitute with VIAL 100 lesa 20 ial standard 400 ul Standards Diluent to make the 200 uM glycerol standard see page 4 for recommended dilution scheme Standards BOTTLE 2 5 ml 4 C pu e Tray Clear polyvinyl tray for multi channel EACH 3 D ee Assay Plates 96 well assay plate blank PLATE 2 sn hae Eel all Other equipment reagents required but not provided with the kit e Single channel pipetter e Multi channel pipetter e Plate reader with a filter of 540 nm e Tubes to dilute glycerol standards e Large gauge needle Rev 04 16 14 Page 2 of 9 PATENT PROTECTED 6 153 432 ASSAY PROCEDURE 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 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 C store any remaining solution refrigerated 2 8 C Bring
8. r 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 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 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 14 Page 7 of 9 PATENT PROTECTED 6 153 432 APPENDIX A PLATE LAYOUT Rev 04 16 14 Page 8 of 9 PATENT PROTECTED 6 153 432 APPENDIX B PROCEDURE 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 well and 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 Mix lysates and transfer 20 ul lysates to the wells containing Wash Bu
9. zenbio gt Triglyceride Assay Kit 100 point assay kit Cat TG 1 NC INSTRUCTION MANUAL ZBMO0013 06 STORAGE CONDITIONS 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 3200 East NC 54 Suite 100 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 14 Page 1 of 9 PATENT PROTECTED 6 153 432 The contents of this kit are sufficient for the assay of 100 assay points in a 96 well plate format The only reagents sold separately are Glycerol Reagent A cat RGTA 10 and the glycerol standard for the Triglyceride Assay kit cat TG GLYSTAN For add
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