InCELL Hunter™ Epigenetic Cell
Contents
1. 6 Once the cells become gt 70 confluent in the T25 flask trypsinize using Cell detachment reagent and transfer the cells to a T75 flask containing 10 12 mLs of cell culture media NOTE To maintain the logarithmic growth of the cells cultures should be maintained in a sub confluent monolayer 7 Passage the cells every 2 3 days based on the doubling time of the cell line using the Cell Detachment Reagent For routine passaging prepare a 1 2 dilution of cells in a total volume of 10 mL of cell culture media Transfer 5 mL of the diluted cells to each of two new T75 flasks containing 10 12 mLs of cell culture media 8 The clone has been found to be stable for at least 10 passages with no significant drop in assay window and ECspo CELL FREEZING PROTOCOL The following procedures are for freezing cells from confluent T225 flasks If smaller flasks are used adjust the volumes accordingly Care should be taken in handling to avoid contamination 1 Remove T225 flasks from incubator and place in tissue culture hood Aspirate the media from the flasks Add 10 mL PBS into each T225 flask and swirl to rinse the cells Aspirate PBS from flask Add 5 mL of Cell Detachment Reagent to the flask Rock the flask back and forth gently to ensure surface of the flask is covered Return flask to the incubator for 5 minutes or until cells have detached Remove the flask from the incubator and view under a microscope to confirm that th2. Cat 92 0018G Series Preserve Freezing Reagent DiscoveRx Cat 92 0017FR Series Cell Detachment Reagent DiscoveRx Cat 92 0009 PathHunter Cell Plating CP Reagent DiscoveRx Cat 93 0563R Series Control compound NOTE Refer target specific data sheet for additional details STORING amp REMOVING CRYOVIALS FROM LIQUID NITROGEN Cryovials are shipped in 2 vials on dry ice and contain 1 2 2 0 x 10 cells vial in 1 mL of freezing medium Upon receipt the vials can be stored for up to 2 weeks at 80 C or transferred to the vapor phase of liquid nitrogen DO NOT store at 80 C for extended periods as this could result in significant loss in cell viability The following procedures are for the safe storage and removal of cryovials from liquid nitrogen storage A face shield gloves and a lab coat should be worn during these procedures 1 InCELL Hunter cells must arrive in a frozen state on dry ice If cells arrive thawed do not proceed contact technical support Frozen cells must be transferred to either liquid nitrogen storage or a 80 C freezer immediately upon arrival If cells will be thawed and used within 24 hours they can be stored temporarily at 80 C For longer storage place vials in the vapor phase of liquid nitrogen storage NOTE CRYOVIALS ARE NOT RATED FOR STORAGE IN THE LIQUID PHASE OF LIQUID NITROGEN CRYOVIALS SHOULD BE STORED IN THE VAPOR PHASE 5 3 When removin
3. 2XL provides a cell based assay to look at protein stability upon compound binding The assay described in this booklet have been validated for use in 384 well microplate formats TECHNOLOGY PRINCI PLE InCELL Hunter cell lines feature a novel in vivo application of the Enzyme Frag ment Complementation EFC technology in which the B galactosidase enzyme has been split into two inactive fragments the enhanced ProLabel ePL and the en zyme acceptor EA The platform measures compound protein binding using a novel B galactosidase tag ePL In this system the protein of interest is tagged to ePL The cellular amount of protein is detected by the addition of enzyme acceptor EA which complements with ePL to form a fully active B galactosidase enzyme that can be quantitatively detected using the chemiluminescent substrate The amount of enzyme activity obtained is proportional to the amount of ePL tagged protein present in the well Cells expressing the designated fusion protein will be tested for changes in protein levels in response to compound treatment ASSAY OVERVIEW To perform InCELL Hunter Assays you will also need the InCELL Hunter Detec tion Kit 96 0002 96 0002L or 96 0002XL in order to generate the chemilumes cent signal Assays should be run using fresh low passage cells that have not been allowed to reach confluency for more than 24 hours Ideally cells should be grown to 70 80 confluence Following cell treatment
4. DiscoverR InCELL Hunter Epigenetic Cell Based Assay For chemiluminescent detection of protein levels User Manual Simple Solutions for Complex Biology CONTENTS LEGAL SECTION INTENDED USE TECHNOLOGY PRINCIPLE ASSAY OVERVIEW MATERIALS PROVIDED MATERIALS NOT PROVIDED REQUIRED STORING amp REMOVING CRYOVIALS FROM LIQUID NITROGEN CELL THAWING AND PROPAGATION METHODS CELL FREEZING PROTOCOL TIPS FOR OPTIMAL PERFORMANCE ASSAY PROCEDURE COMPOUND DOSE RESPONSE CURVE QUICK START PROCEDURE COMPOUND DOSE RESPONSE Read the entire product insert before beginning the assay For additional information or Technical Support contact DiscoveRx or visit www discoverx com PAGE 3 PAGE 4 PAGE 4 PAGE 4 PAGE 4 PAGE 5 PAGE 5 PAGE 6 PAGE 7 PAGE 7 PAGE 8 PAGE 10 LEGAL SECTION This product and or its use is currently subject of pending U S and or foreign patents patent applications The right to use or practice the inventions by using or propagating this product is granted solely in connection with the use of appropriate Detection Reagents protected under trade secret purchased from DiscoveRx Corporation or its authorized distributors LIMITED USE LICENSE AGREEMENT The designated cells and reagents purchased from DiscoveRx are restricted in their use DiscoveRx has developed an assay for Target Engagement InCELL Hunter Assay employing genetically modified cells Cells
5. and detection reagents Reagents collectively referred to as Materials The Cells and Reagents are designed and optimized to be used together in the Assay DiscoveRx wishes to ensure that these Cells and Reagents are used properly and effectively By purchasing the Materials you recognize and agree to the restrictions 1 The Materials cannot be transferred to third parties Transfer to third parties will be permitted only upon written request by Purchaser followed by subsequent written approval by DiscoveRx 2 Purchaser will not analyze the Reagents nor have them analyzed on Purchaser s behalf 3 Purchaser will use only the Reagents supplied by DiscoveRx or an authorized DiscoveRx distributor for the Assays If the purchaser is not willing to accept the limitations of this limited use statement and or has any further questions regarding the rights conferred with purchase of the Materials please contact DiscoveRx Corporation Attn Licensing Department 42501 Albrae Street Fremont CA 94538 tel 510 979 1415 x104 info discoverx com For some products cell lines certain 3 party gene specific patents may be required to use the cell line It is the purchaser s responsibility to determine if such patents or other intellectual property rights are required INTENDED USE InCELL Hunter Epigenetic assay cell Line when used in conjunction with a InCELL Hunter Detection Kit 96 0002 96 0002L or 96 000
6. cells have detached e Add 3 mL of Cell Plating Reagent and transfer to a conical tube Determine cell density using a hemocytometer Using Cell Plating Reagent adjust the volume of the suspension to achieve a cell concentration per well indicated in the target specific data sheet 4 Transfer 20 uL of the cell suspension to each well of a 384 well white walled microplate 5 Incubate the plate overnight at 37 C 5 CO3 Remove InCELL Hunter cells from the incubator previously plated on day 1 Transfer 5 uL of the compound 5X to the plate as shown in the quick start guide Incubate cells with the compounds for the indicated times and temperatures in the target specific data sheet DETECTION REAGENT PREPARATION AND ADDITION 1 4 Prepare InCELL Hunter Detection Reagent as described in the InCELL Hunter Detection Kit Product Insert Cat 96 0002 Add 30 uL of prepared detection reagent to the appropriate wells DO NOT pipette up and down in the well to mix or vortex shake plates a EA reagent Ready to use no preparation necessary b Working Solution Prepare Working Solution by mixing 1 part EA reagent with 1 part Lysis Buffer and 4 parts Substrate Reagent Gently mix the components prior to use Component Entire Plate 384 wells EA Reagent 2 mL Lysis Buffer 2 mL Substrate Reagent 8 mL Incubate for 30 min at room temperature 23 C in the dark before reading the plate on a c
7. e cells are detached Tap the edge of the flask to detach cells from the surface if necessary Add 8 10 mL of Cell Plating Reagent refer target specific data sheet to each T225 flask Rinse the cells from the surface of the flask using the added media Remove the cells from the flask and transfer to a 50 mL conical tube If necessary add an additional 5 mL of reagent to the flask and rinse to collect the remaining cells and transfer the additional volume to the 50 mL conical tube Remove 0 5 mL of the resuspended cells and count the cells using a hemocytometer Centrifuge the collected cells at 1500 x g for 5 minutes After centrifugation discard the supernatant Resuspend the cell pellet in Preserve Freezing Reagent Based on the cell number obtained from Step 5 dilute the resuspended cells to a concentration of 1 2 2 0 x 10 cells mL Transfer 1 mL cells to each 2 mL cryogenic tube Keep cells on ice during this process and transfer to a cryogenic container pre chilled at 4 C Transfer tubes to 80 C and store overnight Transfer tubes into the vapor phase of a liquid nitrogen tank for long term storage TIPS FOR OPTIMAL PERFORMANCE Cells must be maintained exactly as mentioned to maintain expression of fusion protein Ideally cells should be maintained at approximately 70 confluence Cells should not be allowed to grow at confluence for more than 24 hours Allowing the cells to adhere and grow overnight prior to an
8. g cryovials from liquid N2 storage use tongs and place immediately on dry ice in a covered container Wait at least one minute for any liquid nitrogen inside the vial to evaporate 4 Proceed with the thawing protocol in the following section CELL THAWING AND PROPAGATION METHODS The following procedures are for thawing cells in cryovials seeding and expanding the cells and maintaining the cultures once the cells are expanded Cells are free of contamination prior to shipment and care should be taken in their handling to avoid contaminating them Face shield gloves and a lab coat should be worn during the thawing procedure Pre warm 5 10 mL Revive Medium RM in a 37 C water bath 2 Place the frozen cell vials briefly in a 37 C water bath under sterile conditions until only small ice crystals remain and the cell pellet is almost completely thawed 30 sec 1 min Caution Longer incubation times may result in cell death 3 Transfer thawed cells to a sterile 15 mL conical tube containing the 5 10 mL of pre warmed Revive medium Centrifuge at 300 x g for 4 minutes to pellet cells Remove media 4 Resuspend cell pellet in 5 mLs of pre warmed RM Transfer cells to a T25 flask and incubate for 24 hours at 37 C 5 CO2 5 After 24 hours gently remove media being careful not to disturb the cell monolayer and replace with 5 mLs of Cell Culture Media refer to target specific data sheet for specific Cell Culture Media requirements
9. hemiluminescent reader NOTE For a list of readers and settings please visit the URL below http www discoverx com instrument_chart php Read samples on any standard luminescence plate reader Compound potencies can be derived from a four parameter nonlinear curve fitting analysis Use GraphPad Prism or other comparable program to plot your compound dose response QUICK START PROCEDURE COMPOUND DOSE RESPONSE In a white walled 384 well plate perform the following Seed cells using 20 pL Cell Plating Reagent Refer to target specific data sheet for the cell numbers per well Incubate cells overnight 37 C Induce cells with 5 uL compound 5X Incubate for indicated time and temperature refer target specific data sheet Add 30 uL InCELL Hunter Detection Reagent Incubate for 30 min RT Read Chemiluminescent Signal 10 NOTES 11 Contact I nformation DiscoveRx Corporation World Wide Headquarters 42501 Albrae Street Fremont CA 94538 United States t 510 979 1415 f 510 979 1650 toll free 866 448 4864 KINOMEscan A division of DiscoveRx 11180 Roselle Street Suite D San Diego CA 92121 United States 00 644 5687 58 630 4600 00 tl f DiscoveRx Corporation Ltd Europe Headquarters Faraday Wharf Holt Street Aston Science Park Birmingham B7 4BB United Kingdom t 44 121 260 6142 f 44 121 260 6143 ww
10. the assay is performed by adding a working solution of InCELL Hunter Detection Reagents to the treated cells in a no mix one addition protocol After addition of the detection reagents the samples must be read after 30 minutes The Assay Procedure sections and Quick Start Guide in this booklet contain detailed information about how to run the assay MATERIALS PROVIDED Description Storage InCELL Hunter cells 2 vials Liquid N2 Vapor phase NOTE Please refer to the datasheet of the InCELL Hunter cell line for detailed information on the target that you are testing MATERIALS NOT PROVIDED REQUIRED The following equipment and additional materials are required to perform InCELL Hunter Assays Equipment Materials Single and multichannel micro pipettors and pipette tips Tissue culture disposables and plasticware T25 and T75 flasks etc Cryogenic vials for freezing cells V bottom 384 well compound dilution plates DiscoveRx Cat 92 0011 or similar Disposable Reagent Reservoir Thermo Scientific Cat 8094 or similar Hemocytometer White wall clear bottom 384 well microplates DiscoveRx Cat 92 0013 or similar Multimode or luminescence plate reader LumiLite DiscoveRx Cat 75 0001 or similar InCELL Hunter Detection Kit DiscoveRx Cat 96 0002 series Revive Media DiscoveRx Cat 92 0016RM Series PathHunter select Cell Culture Kits DiscoveRx
11. w discoverx com Discover DRX UM InCELL HUNTER EPIGENETIC 1012V1 2012 DiscoveRx Corporation Fremont CA 94538 All rights reserved
12. y assay is recommended ASSAY PROCEDURE COMPOUND DOSE RESPONSE CURVE The steps outlined below provide the assay volumes and procedures for performing assays using the InCELL Hunter Cell Lines and InCELL Hunter Detection Reagents in a 384 well format Although plate layouts and experimental designs may vary we recommend performing a 12 point dose curve for each compound using at least duplicate Compound 1 Compound 3 Compound 5 Compound 7 Compound 9 Compound 11 Compound 13 p N Compound 15 o Figure 1 wells for each dilution 3 fold serial 2 3 fold serial Low dilutions of compound High lt Low dilutions of compound High 2 3 45 6 7 8 9 1011 12 13 14 15 16 17 18 19 20 21 22 23 24 No Compound No Compound 1 A Compound 2 B Compound 4 Compound 6 F G C d 8 H Ompoun l Compound 10 J Compound 12 Compound 14 Compound 16 This plate map shows 12 point dose curves with 2 data points at each concentration for 16 compounds per plate for a total of 160 compound dilutions per 384 well plate PROTOCOL COMPOUND DOSE RESPONSE 1 Harvest cells as follows from a confluent T25 or T75 flask using Cell Detachment Reagent DiscoveRx Cat 92 0009 a Remove medium b Wash cells with 5 mL PBS and aspirate c Add 0 5 mL Cell Detachment Reagent for a T25 flask or 1 mL Cell Detach ment Reagent for a T75 flask d Place flask in the incubator for 5 minutes or until
Download Pdf Manuals
Related Search