User Manual - Biomol GmbH
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1. 4 ul 400 ng Cignal negative control 4 volumes for conditions 5 to 8 of Table 5 for every well dilute 1 ul 100 ng of Cignal reporter in 25 ul of Opti MEM serum free culture medium Tube 9 25 ul Opti MEM 1ul 100 ng Cignal positive control Mix each transfection cocktail gently 2 Prepare a SureFECT dilution for 9 tubes mentioned in step 1 by dispensing 2 7 ul of SureFECT into 225 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 100 ul of diluted SureFECT to the two tubes mentioned in step 1 containing equal volume 100 ul of diluted Cignal reporter and add 25 ul of diluted SureFECT into the positive control tube containing 25 ul of diluted constructs 1 1 ratio as detailed in Table 5 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2x10 cells ml in Opti MEM containing 5 of fetal bovine serum a2. Expression of the Monster GFP reporter can be monitored via FACS flow cytometry fluorescent microscopy or standard fluorometry The spectral properties of the Monster Green Fluorescent Protein are slightly red shifted compared to other commercially available GFP reporters We recommend using the standard FACS settings of an argon laser 488nm excitation and filters of 530 15 nm 530 30nm for emission When analyzing GFP expression via fluorescent microscopy or standard fluorometry we recommend using standard fluoroisothiocyanate FITC filters excitation of 470 20nm and an emission filter of 515nm long pass We found some components in DMEM interfere with the SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that had been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support support SABiosciences com www SABiosciences com 27 G Transfection and Treatment Protocol for Reporter Assay Peptide Recombinant Protein The following protocol is designed to reverse transfect adherent cell line HEK 293H using SureF
3. In addition most consensus sequences for transcription factor binding have been removed from the synthetic GFP gene in order to minimize aberrant transcription and improve the reliability of the GFP as an accurate reporter The spectral properties of the synthetic GFP are slightly red shifted compared to other commercially available GFPs Peak excitation occurs at 505nm with a shoulder at 480nm peak emission occurs at 515nm Technical Support 888 503 3187 US 301 682 9200 Version 1 6 Benefits of Cignal Reporter Assays e PERFORMANCE Both the dual luciferase and GFP reporter systems provide exceptional sensitivity reproducibility specificity and signal to noise ratio e VERSATILITY Can monitor signal transduction pathway activity utilizing the dual luciferase reporter system in an endpoint format assay or measure pathway activation dynamics on live cells using the GFP reporter system e CONVENIENCE Transfection ready constructs including positive and negative controls coupled with a transient reporter system enable rapid analysis of signal transduction pathway regulation Technical Support support SABiosciences com www SABiosciences com 5 How CIGNAL REPORTER LUC ASSAYS WORK actor Control Control Q Reporter Transcription Negative Positive 5 Monster GFP Firefly Luciferase Firefly Luciferase a l Tendem TRE Firefly Luciferase Renilla Luciferase Figure 1A Overview of Cignal Dual Luci
4. Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately measure the cell density with a hemacytometer or an automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific constructs sIRNA SureFECT complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 2 x 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing constructs siIRNA SureFECT complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth 8 Incubate cells at 37 C in a 5 CO incubator for 16 24 hours 9 After 16 24 hours of transfection change the medium to complete growth medium DMEM with 10 FBS 0 1mM NEAA 1mM Sodium pyruvate 100 U ml penicillin and 100 ug ml streptomycin 10 To study the effect of knockdown we recommend harvesting cells 48 or 72 hours after transfection to perform dual luciferase assay 12 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay Expression of the Monster GFP reporter can be monitored via FACS flow cytome
5. Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SABiosciences Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product NOTICE TO PURCHASER This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use The purchase of Cignal Reporter Assay kits includes a limited nonexclusive license to use the kit components for research use only This license does not grant rights to use the kit components for reproduction of any constructs to modify kit components for resale or to manufacture commercial products without written approval of SABiosciences Corporation No other license expressed implied or by estoppel is granted NOTICE TO PURCHASER II The Dual Luciferase Reporter Assay and Monster Green Fluorescent Protein are trademarks of Promega Corporation SABiosciences Corporation 6951 Executive Way Suite 100 Frederick MD 21703 USA CONTENTS l Introduction 4 II Product Contents and Descriptions 8 III Additional Materials Required 12 IV Protocol 13 A Before you begin 13 B Generalized Transfection Protocols 14 C Co transfection Protocol for siRNA Reporter Assay 16 D Co transfection Protocol for shRNA Reporter Assay 19 E Co tra
6. ul 25 ul 100 ng 3 1 0 ul 200 ng 25 ul 0 3 ul 25 ul 48 hor 72h 100 ng 4 1 0 ul 200 ng 25 ul 0 3 ul 25 ul 100 ng 5 1 0 ul 25 ul 0 3 ul 25 ul 1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 Add 25 ul of Opti MEM to each of 5 polystyrene tubes avoid using DMEM along with the following Experimental transfection 1 ul 100 ng Cignal reporter 200 ng sequence specific shRNA Control transfections 1 ul 100 ng Cignal reporter 200 ng negative control snRNA 1 ul 100 ng Cignal negative control 200 ng sequence specific shRNA 1 ul 100 ng Cignal negative control 200 ng negative control shRNA Technical Support support SABiosciences com www SABiosciences com 19 1 ul 100 ng Cignal positive control Mix each transfection cocktail gently 2 Prepare a SureFECT dilution for 5 tubes mentioned in step 1 by dispensing 1 5 ul of SureFECT into 125 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polysty
7. 25 ul 4 a 100x 25 ul 0 3 pl 25 ul 5 a fi 25 ul 0 3 ul 25 ul 30 hor 42h 6 a a 1x 25 ul 0 3 ul 25 ul 7 a F 10x 25 ul 0 3 ul 25 ul 8 Ac an 100x 25 ul 0 3 ul 25 ul 9 as A 25 ul 0 3 ul 25 ul 4X is a smallest appropriate amount of interfering peptide recombinant protein growth factor expected to modulate signaling pathway _1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 Technical Support 888 503 3187 US 301 682 9200 Version 1 6 Set up three polystyrene tubes as follows Experimental transfections Tubes 1 4 100 pl Opti MEM 4 ul 400 ng Cignal reporter 4 volumes for conditions 1 to 4 of Table 6 for every well dilute 1 ul 100 ng of Cignal reporter in 25 ul of Opti MEM serum free culture medium Control transfections Tubes 5 8 100 ul Opti MEM 4 ul 400 ng Cignal negative control 4 volumes for conditions 5 to 8 of Table 6 for every well dilute 1 ul 100 ng of Cignal reporter in 25 ul of Opti MEM serum free culture medium Tube 9 25 ul Opti MEM 1ul 100 ng Cignal positi
8. 8 TGFB SMAD2 SMAD3 SMAD4 CCS 017L CCS 017G Vitamin D VDR CCS 2029L Wnt TCF LEF CCS 018L CCS 018G Xenobiotic AhR CCS 2045L Cignal Reporter Assay Controls Control Construct Components Concentration Catalog Number And Volume A mixture of non inducible Cignal Negative Control firefly luciferase reporter 100 ng ul 500 ul CCS NCL LUC construct and constitutively expressing Renilla luciferase construct 40 1 A mixture of a constitutively Cignal Positive Control expressing GFP construct 100 ng ul 250 pl CCS PCL LUC constitutively expressing firefly luciferase construct and constitutively expressing Renilla Technical Support support SABiosciences com www SABiosciences com 33 luciferase element 40 1 1 Cignal Negative Control A GFP reporter construct in GFP which GFP expression is 100 ng ul 500 ul CCS NCG controlled by a minimal promoter Cignal Positive Control A constitutively expressing 100 ng ul 250 ul CCS PCG GFP GFP construct Notes Technical Support 888 503 3187 US 301 682 9200 Notes Cignal Reporter Assay Kits Part 1030A Version 1 6 11 20 2009 fy SABiosciences Focus on Your Pathway BIOMOL GmbH Waidmannstr 35 O 22769 Hamburg b m info biomol de 10 o www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D 888 503 3187 301 682 9200 www SABiosciences com support SABioscien
9. A SABiosciences BIOMOL GmbH Waidmannstr 35 22769 Hamburg O biomol info biomol de www biomol de S e r a n u a Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D E Cignal Reporter Assay Kits Cell Based Pathway Activity Assays See Purchaser Notification for limited use license and warranty information pages 2 and 3 Part 1030A Version 1 6 11 20 2009 SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA Cignal Reporter Assay Kits Cell Based Pathway Activity Assays User Manual For Catalog Numbers CCS L and CCS G Ordering and Technical Service Contact Information e Tel 1 888 503 3187 US 301 682 9200 outside US e Fax 1 888 465 9859 US 301 682 7300 outside US e On line Order www SABiosciences com e E MAIL order SABiosciences com to place an order support SABiosciences com for technical support You may place orders by fax e mail or from our website Each order should include the following information Your contact information name phone email address Product name catalog number and quantity Purchase order number or credit card information Visa or MasterCard Shipping address Billing address For more information visit us at www SABiosciences com LIMITED PRODUCT WARRANTY This warranty limits our liability to replace this product in the event the product fails to perform due to any manufacturing defect SABiosciences
10. ECT Transfection Reagent Cat No SA 01 in a 96 well plate format The Cignal reporter assay works well with transfection reagent from other vendors f you are using transfection reagent other than SureFECT follow their manufacturer s protocol for transfection The Cignal Reporter Assay also works well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the component in proportion to the surface area see section IV F This is just a general quideline the optimal conditions amounts should be adjusted according to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 6 Guidelines for studying the effect of a peptide or recombinant protein Table 6 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol Cignal Cignal Cignal Peptide or Opti MEM SureFECT Opti MEM Time of Reporter Negative Positive Recombinant DNA Diluent per well SureFECT Transfection per well Control Control Protein per well diluent hours per well per well per well per well 1 A 25 ul 0 3 ul 25 ul 2 i a 1x 25 ul 0 3 ul 25 ul 3 Y 0 10x 25 ul 0 3 pl
11. S without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2x10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately measure the cell density with a hemacytometer or automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific complexes into the appropriate wells Technical Support support SABiosciences com www SABiosciences com 23 7 Add 100 ul of prepared cell suspension 2x10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing construct vector SureFECT complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth 8 Incubate cells at 37 C in a 5 CO incubator for 16 24 hours 9 After 16 24 hours of transfection change the medium to complete growth medium DMEM with 10 FBS 0 1mM NEAA 1mM Sodium pyruvate 100 U ml penicillin and 100 ug ml streptomycin 11 To study the effect of the gene product we recommend harvesting cells 32 hours or 48 hours after transfection to perform the dual luciferase assay 12 The luciferase assay can be developed by using Dual Lucifer
12. SA 01 in a 96 well plate format The Cignal Reporter Assay works well with transfection reagent from other vendors If you are using transfection reagent other than SureFECT follow their manufacturer s protocol for transfection The Cignal Reporter Assay also works well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the components in proportion to the surface area see section IV H This is just_a general quideline the optimal conditions amounts should be adjusted according to the cell type and study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 3 Guidelines for setting up co transfections of a shRNA vector and Cignal Reporter Assay Table 3 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol Cignal Cignal Cignal Specific Negative Opti MEM SureFECT Opti MEM Time of Reporter Negative Positive shRNA Control Nucleic per well SureFECT transfection per well Control Control per well shRNA Acid Diluent hours per well per well per well Diluent per well per well 100 ng 1 1 0 ul 200 ng 25 ul 0 3 ul 25 ul 100 ng 2 1 0 ul 200 ng 25 ul 0 3
13. ase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay Expression of the Monster GFP reporter can be monitored via FACS flow cytometry fluorescent microscopy or standard fluorometry The spectral properties of the Monster Green Fluorescent Protein are slightly red shifted compared to other commercially available GFP reporters We recommend using the standard FACS settings of an argon laser 488nm excitation and filters of 530 15 nm 530 30nm for emission When analyzing GFP expression via fluorescent microscopy or standard fluorometry we recommend using standard fluoroisothiocyanate FITC filters excitation of 470 20nm and an emission filter of 515nm long pass We have found that some components in DMEM interfere with SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that have been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support 888 503 3187 US 301 682 9200 Version 1 6 F Transfection and Treatment Protocol for Reporter Assay Small Molecu
14. ces com Technical Support 888 503 3187 US 301 682 9200
15. construct Figure 2D pre mixed with a constitutively expressing firefly luciferase construct Figure 2E and a constitutively expressing Renilla luciferase construct Figure 2B 40 1 1 The positive control is necessary for visual confirmation of transfection It is also useful for transfection optimization studies The expression of the GFP from the positive control construct can be monitored by fluorescence microscopy using an excitation filter of 470 20 nm 470 40 nm and an emission filter of 515 nm long pass Tandem repeats TATA box A of TREs y Firefly Luc B n CMV immediate early enhancer promoter TATA box C Firefly Luc I CMV immediate early m enhancer promoter E eee Firefly Luc Figure 2 Schematic representation of constructs involved in the Cignal Reporter Assay A The inducible transcription factor responsive construct expressing firefly luciferase B The constitutively expressing Renilla luciferase construct C The non inducible firefly luciferase reporter construct D The constitutively expressing GFP construct and E The constitutively expressing firefly luciferase construct Technical Support support SABiosciences com www SABiosciences com g B GFP Reporter Assay Kits 1 Kit Contents Table 2 Cignal GFP Reporter Assay Kit Specifications Component Specification Concentration and total volume An inducible transcription factor responsive R
16. ction of proteins peptides ligands and small molecule compounds Each of the dual luciferase formatted reporters encodes for the mammalian codon optimized non secreted form of the firefly luciferase gene carrying a protein destabilizing sequence Cells rapidly degrade the destabilized form of the firefly luciferase protein and hence the background luciferase activity noise level is greatly reduced Due to low background activity the magnitude of the response that can be measured signal to noise ratio as well as the speed of measuring changes in transcription are enhanced The Cignal dual luciferase reporter assays provide outstanding reproducibility sensitivity specificity and signal to noise ratio They are extremely useful for carrying out endpoint pathway regulation assays The Cignal GFP reporter assays enable you to monitor the dynamics of pathway activation on living cells with single cell resolution The Cignal GFP reporter constructs utilize the Monster Green Fluorescent Protein reporter gene Monster GFP is encoded by an improved synthetic version of the green fluorescent protein gene This GFP expression cassette has been codon optimized to maximize mammalian cell expression and also utilizes an optimized Kozak sequence to increase translation efficiency The synthetic GFP is an ideal fluorescent reporter providing high level fluorescence and minimal cytotoxicity Moreover the synthetic GFP gene is resistant to photobleaching
17. ell diluent hours per well Construct Compound per well per well per well 100 ng 1 1 0 ul 25 ul 0 3 ul 25 ul 100 ng a 2 1 0 ul 1X 25 ul 0 3 ul 25 ul 100 ng 3 1 0 ul 10X 25 ul 0 3 pl 25 ul 100 ng 4 1 0 ul 100X 25 ul 0 3 ul 25 ul 100 ng 5 1 0 ul 25 ul 0 3 pl 25 ul 30 hor 42h 100 ng 6 1 0 ul 1X 25 ul 0 3 ul 25 ul 100 ng 7 1 0 ul 10X 25 ul 0 3 ul 25 ul 100 ng 8 1 0 ul 100X 25 ul 0 3 ul 25 ul 100 ng 9 1 0 ul 25 ul 0 3 ul 25 ul 1X is a smallest appropriate amount of small molecule or organic compound expected to modulate signaling pathway 1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 Technical Support support SABiosciences com www SABiosciences com 25 Set up three polystyrene tubes as follows Experimental transfections Tubes 1 4 100 ul Opti MEM 4 ul 400 ng Cignal reporter 4 volumes for conditions 1 to 4 of Table 5 for every well dilute 1 ul 100 ng of Cignal reporter in 25 ul of Opti MEM serum free culture medium Control transfections Tubes 5 8 100 ul Opti MEM
18. eporter GFP reporter 100 ng ul 500 pl Negative A GFP reporter construct in which GFP control expression is controlled by a minimal 100 ng ul 500 ul promoter Positive A constitutively expressing GFP construct control 100 ng ul 250 ul NOTE These constructs are transfection grade and are ready for transient transfection These constructs are specifically designed to inhibit transformation and are NOT MEANT for introduction and amplification in bacteria 2 Description Each Cignal GFP Reporter Assay Kit includes the following components 1 Reporter The inducible transcription factor responsive GFP reporter encodes the green fluorescent protein gene under the control of a basal promoter element TATA box joined to tandem repeats of a specific Transcriptional Response Element TRE Figure 3A This construct monitors both increases and decreases in the activity of a key transcription factor which is a downstream target of a specific signaling pathway Negative control The negative control is a GFP reporter that encodes the green fluorescent protein under the control of a basal promoter element TATA box without any additional transcriptional response elements Figure 3B The negative control is critical to identifying pathway specific effects and determining background reporter activity Positive control The positive control is a constitutively expressing GFP construct Figure 3C The positive control is necessa
19. erest Table 7 Reagent amounts for transfecting cells in different size culture vessels Type of Surface Starting Starting Volume of Starting Volume of siRNA shRNA Plate Area amount of Volume of Cell No of Opti MEM Vector or Gene cm per construct SureFECT Suspension Adherent Medium Expression Vector well ng well ul well ul well Cells per ul per Well Well 96 well 0 3 100 0 3 100 20 000 2 X 25 2 pmol 200 ng 48 well 0 95 150 0 8 250 62 500 2X50 5 pmol 500 ng 24 well 1 9 250 1 6 500 125 000 2X50 10 pmol 750 ng 12 well 3 8 500 3 2 1000 250 000 2 X100 20 pmol 1 5 ug 6 well 9 4 1000 8 0 2500 750 000 2 X 250 50 pmol 4 0 pg 35 mm 8 0 1000 8 0 2500 750 000 2 X 250 50 pmol 4 0 pg 6 60mm 21 2000 18 0 5000 1 5X 10 2 X 500 100 pmol 8 0 pg 6 100mm 55 5000 45 0 15000 15ml 4 5X 10 2 X 1500 300 pmol 25 ug a 2X means one volume of Opti MEM medium for diluting constructs and another volume of Opti MEM medium for diluting SureFECT For any other troubleshooting or technical questions about the Cignal Reporter Assay please call one of our Technical Support representatives at 1 888 503 3187 or 301 682 9200 or email at support SABiosciences com Technical Support support SABiosciences com www SABiosciences com 31 Appendix Cignal Reporter Assay Kits Dual luciferase Cat No MGFP Pathway Tra
20. esponsive construct and constitutively expressing Renilla luciferase construct 40 1 The inducible transcription factor responsive construct encodes the firefly luciferase reporter gene under the control of a basal promoter element TATA box joined to tandem repeats of a specific Transcriptional Response Element TRE Figure 2A This construct monitors both increases and decreases in the activity of a key transcription factor which is a downstream target of a specific signaling pathway The constitutively expressing Renilla construct encodes the Renilla luciferase reporter gene under the control of a CMV immediate early enhancer promoter Figure 2B and acts as an internal control for normalizing transfection efficiencies and monitoring cell viability It is also useful to confirm transfection and to verify active luciferase in the transfected culture Negative control The negative control is a mixture of non inducible reporter construct and constitutively expressing Renilla luciferase construct 40 1 The non inducible Technical Support 888 503 3187 US 301 682 9200 Version 1 6 reporter construct encodes firefly luciferase under the control of a basal promoter element TATA box without any additional transcriptional response elements Figure 2C The negative control is critical to identifying specific effects and determining background reporter activity Positive control The positive control is a constitutively expressing GFP
21. ferase Reporter Assays Process Technical Support 888 503 3187 US 301 682 9200 Version 1 6 How CIGNAL REPORTER GFP ASSAYS WORK Figure 1B Overview of Cignal GFP Reporter Assays Process Technical Support support SABiosciences com www SABiosciences com 7 ll Product Contents and Descriptions A Dual Luciferase Reporter Assay Kits 1 Kit Contents Table 1 Cignal Reporter Assay Kit Specifications Component Specification Concentration and total volume A mixture of an inducible transcription factor Reporter responsive firefly luciferase reporter and 100 ng ul 500 ul constitutively expressing Renilla construct 40 1 Negative A mixture of non inducible firefly luciferase control reporter and constitutively expressing 100 ng ul 500 ul Renilla construct 40 1 control luciferase construct and constitutively A mixture of a constitutively expressing GFP Positive construct constitutively expressing firefly 100 ng ul 250 ul expressing Renilla luciferase construct 40 1 1 NOTE These constructs are transfection grade and are ready for transient transfection These constructs are specifically designed to inhibit transformation and are NOT MEANT for introduction and amplification in bacteria 2 Description Each Cignal Reporter Assay Kit includes the following components 1 Reporter The reporter is a mixture of inducible transcription factor r
22. ime of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support support SABiosciences com www SABiosciences com 21 E Co transfection Protocol for Expression Vector Reporter Assay The following protocol is designed to reverse transfect adherent cell line HEK 293H using SureFECT Transfection Reagent Cat No SA 01 in a 96 well plate format The Cignal Reporter Assay works well with transfection reagent from other vendors If you are using transfection reagent other than SureFECT follow their manufacturer s protocol for transfection The Cignal Reporter Assay also works well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the component in proportion to the surface area see section IV H This is just a general quideline the optimal conditions amounts should be adjusted according to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 4 Guidelines for setting up co transfections of an expression vector and Cignal Reporter Assay Table 4 represents the
23. ing to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 2 Guidelines for setting up co transfections of siRNA and Cignal Reporter Assays Table 2 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol Cignal Cignal Cignal Specific Negative Opti MEM SureFECT Opti MEM Time of Reporter Negative Positive siRNA Control Nucleic per well SureFECT transfection per well Control Control per well siRNA Acid Diluent hours per well per well per well Diluent per well per well 100 ng 1 1 0 nl 2 pmol 25 ul 0 3 ul 25 ul 100 ng 2 1 0 ul 2 pmol 25 ul 0 3 ul 25 ul 100 ng 3 1 0 ul 2 pmol 25 ul 0 3 ul 25 ul 48 hor 72h 100 ng 4 1 0 ul 2 pmol 25 ul 0 3 ul 25 ul 100 ng 5 1 0 ul 25 ul 0 3 ul 25 ul 1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compen
24. injectors 96 well white opaque flat bottom microtiter plate Luminometer FACS flow cytometer fluorescent microscope or fluorometer Technical Support 888 503 3187 US 301 682 9200 Version 1 6 IV Protocol A Before you begin 1 Cell line selection The Cignal Reporter Assay may be used with various mammalian cell lines Cell lines show a great deal of variation in the levels of signaling proteins The transcriptional activator activities in the cell line used will determine the sensitivity of the assay A cell line should be selected based on the functionality of the signal transduction pathway under investigation as well as for the transfectability of the cell line see below Transfection reagent selection We recommend the use of SureFECT SABiosciences Cat No SA 01 as a transfection reagent The Cignal Reporter Assay however also performs equally well with other transfection reagents such as Lipofectamine 2000 Invitrogen Cat No 11668 027 or FUGENE 6 Roche Cat No 1815091 When using alternative transfection reagents please refer to the manufacturer s instructions on the use of those reagents Optimization of transfection conditions The sensitivity of the Cignal Reporter Assay depends on the transfection efficiency The transfection efficiency in turn primarily depends upon cell line used Therefore it is very important to optimize the transfection conditions for each cell type under study Va
25. les Organic Compounds The following protocol is designed to reverse transfect adherent cell line HEK 293H using SureFECT SABiosciences Cat No SA 01 as a transfection reagent in 96 well plate format The Cignal reporter assay works well with transfection reagent from other vendors If you are using transfection reagent other than SureFECT follow their manufacturer s protocol for transfection The Cignal Reporter Assay also works well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the component in proportion to the surface area see section IV H This is just a general guideline the optimal conditions amounts should be adjusted according to the cell type and the study requirements Read the protocol completely before starting the experiment IMPORTANT 1 Do not add antibiotics to media during transfection as this causes cell death 2 Avoid the use of DMEM medium Table 5 Guidelines for studying the effect of small molecules organic compounds Table 5 represents the total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol Cignal Cignal Cignal Small Opti MEM SureFECT Opti MEM Time of Reporter Negative Positive Molecule DNA diluent per well SureFECT Transfection per well Control Control Organic per w
26. n change the medium to complete growth medium DMEM with 10 FBS 0 1mM NEAA 1mM Sodium pyruvate 100 U ml penicillin and 100 ug ml streptomycin 10 To study the effect of knockdown we recommend harvesting cells 48 or 72 hours after transfection to perform dual luciferase assay 11 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay Expression of the Monster GFP reporter can be monitored via FACS flow cytometry fluorescent microscopy or standard fluorometry The spectral properties of the Monster Green Fluorescent Protein are slightly red shifted compared to other commercially available GFP reporters We recommend using the standard FACS settings of an argon laser 488nm excitation and filters of 530 15 nm 530 30nm for emission When analyzing GFP expression via fluorescent microscopy or standard fluorometry we recommend using standard fluoroisothiocyanate FITC filters excitation of 470 20nm and an emission filter of 515nm long pass We have found that some components in DMEM interfere with SureFECT transfection Technical Support 888 503 3187 US 301 682 9200 Version 1 6 protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that have been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the t
27. nd 1 NEAA To ensure reproducible transfection results it is important to accurately determine the cell density with a hemacytometer or an automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 2 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing constructs SureFECT complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth 8 Incubate cells at 37 C in a 5 COs incubator for 16 hours 9 After 16 hours of transfection change medium to assay medium Opti MEM containing 0 5 of fetal bovine serum 1 NEAA 100 U ml Penicillin and 100 ug ml Streptomycin 10 After 24 hours of transfection treat the cells as described in Table 5 with 1x 10x and 100x amount of small molecule or organic compound 1x is the lowest appropriate amount of small molecule or organic compound expected to modulate the signaling pathway Technical Support 888 503 3187 US 301 682 9200 Version 1 6 11 To study the effect of small molecule or organic compound we recommend harvesting cells 6 hours or 18 hours after treatment to perform dual luciferase assay 12 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay
28. nscription Factor Cat No Amino Acid Deprivation ATF4 ATF3 ATF2 CCS 8034L Androgen Androgen Receptor CCS 1019L Antioxidant Response Nrf2 amp Nrf1 CCS 5020L ATF6 ATF6 CCS 8034L C EBP C EBP CCS 001L cAMP PKA CREB CCS 002L CCS 002G Cell Cycle E2F DP1 CCS 003L DNA Damage p53 CCS 004L Early Growth Response EGR1 CCS 8021L ER Stress CBF NF Y YY1 CCS 2032L Estrogen Receptor Estrogen Receptor ER CCS 005L GATA GATA CCS 1035L Glucocorticoid Receptor Glucocorticoid Receptor GR CCS 006L Heat Shock Response HSF CCS 4023L Heavy Metal Response MTF1 CCS 5033L Hedgehog Gli CCS 6030L Hepatocyte Nuclear Factor 4 HNF4 CCS 3039L Hypoxia HIF 1 CCS 007L Interferon Regulation IRF 1 CCS 7040L Interferon Type STAT1 STAT2 CCS 008L Interferon Gamma STAT1 STAT1 CCS 009L KLF4 KLF4 CCS 4036L Liver X Receptor LXRa CCS 0041L MAPK ERK Elk 1 SRF CCS 010L CCS 010G MAPK JNK AP 1 CCS 011L CCS 011G Technical Support 888 503 3187 US 301 682 9200 Version 1 6 MEF2 MEF2 CCS 7024L c Myc Myc Max CCS 012L Nanog Nanog CCS 7037L NFkB NFkB CCS 013L CCS 013G Notch RBP J CCS 014L Oct4 Oct4 CCS 0025L Pax6 Pax6 CCS 3042L PI3K AKT FOXO CCS 1022L PKC Ca NFAT CCS 015L PPAR PPAR CCS 3026L Progesterone Progesterone Receptor CCS 6043L Retinoic Acid Receptor RAR CCS 016L Retinoid X Receptor RXR CCS 9044L Sox2 Sox2 CCS 0038L SP1 SP1 CCS 6027L STAT3 STAT3 CCS 902
29. nsfection Protocol for Expression Vector Reporter Assay 22 F Transfection and Treatment Protocol for Reporter Assay 25 Small Molecules Organic Compounds G Transfection and Treatment Protocol for Reporter Assay 28 Peptide Recombinant Protein H Scaling up Transfection Experiments 31 Appendix Cignal Reporter Assay Kits and Controls 32 Technical Support support SABiosciences com www SABiosciences com 3 Introduction The Cignal Reporter Assays from SABiosciences are designed for accurate sensitive and quantitative assessment of the activation of signal transduction pathways SABiosciences has developed a series of inducible reporter constructs that encode a reporter gene under the control of a basal promoter element TATA box joined to tandem repeats of specific transcriptional response elements TRE Figure 1 Transcription factor activities can act as readouts for the intracellular status of many signal transduction pathways Our constructs are specifically engineered for measuring changes in activity both increases and decreases of these signaling pathways Each of the Cignal reporter assays is available in a dual luciferase format In addition six of the Cignal reporter assays are also available as GFP reporter constructs These include the CRE SRE AP 1 NFkB SMAD and TCF LEF Cignal Reporter Assays The Cignal reporter assays are valuable tools for deciphering gene function as well as determining the mechanism of a
30. nterest 100 ng carrier DNA 1 ul 100 ng Cignal reporter 200 ng experimental vector expressing gene of interest Control transfections 1 ul 100 ng Cignal reporter 100 ng negative control expression vector 100 ng carrier DNA 1 ul 100 ng Cignal reporter 200 ng negative control expression vector 1 ul 100 ng Cignal negative control 100 ng experimental vector expressing gene of interest 100 ng carrier DNA 1 ul 100 ng Cignal negative control 200 ng experimental vector expressing gene of interest 1 ul 100 ng Cignal negative control 100 ng negative control expression vector 100 ng carrier DNA 1 ul 100 ng Cignal negative control 200 ng negative control expression vector 1 ul 100 ng Cignal positive control Mix each transfection cocktail gently 2 Prepare a SureFECT dilution for 9 tubes mentioned in step 1 by dispensing 2 7 ul of SureFECT into 225 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 25 ul of diluted SureFECT into each of the nine tubes containing 25 ul of diluted constructs 1 1 ratio as detailed in Table 4 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in a culture dish once with Dulbecco s PB
31. ntrol for test nucleic acid iii Cignal Negative Control test nucleic acid expression plasmids shRNA plasmids or siRNAs v Cignal Negative Control negative control for test nucleic acid Cignal Positive Control 7 Dilute SureFECT into Opti MEM Add diluted SureFECT to nucleic acid mixtures incubate at room temperature for 20 minutes Trypsinize if necessary count and suspend cells to appropriate density Aliquot transfection complexes into wells Immediately seed cells to each well Technical Support 888 503 3187 US 301 682 9200 Version 1 6 For detailed information on the transfection conditions and treatment of cultures post transfection refer to the application specific protocols within this user manual 2 Traditional Transfection Protocol Overview 2 DAY PROCEDURE Traditional Transfection DAY 1 Add Cells and Medium 900 aE 96 well Cell Culture Plate LEE Add SureFECT Transfection Reagent Cignal Reporter and Test Nucleic Acids DAY 1 Trypsinize if necessary count and suspend cells to appropriate density Seed cells into multiwell plate s DAY 2 Prepare nucleic acid mixtures in appropriate ratios This may include any of the following combinations depending upon the experimental design we recommend carrying out each transfection condition in triplicate Experimental transfection i Cignal Reporter test nucleic acid expression plasmids shRNA plasmids or
32. ot 50 ul of specific complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 2 10 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing constructs SureFECT complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth 8 Incubate cells at 37 C in a 5 CQOz incubator for 16 hours 9 After 16 hours of transfection change medium to assay medium Opti MEM containing 0 5 of fetal bovine serum 1 NEAA 100 U ml Penicillin and 100 ug ml Streptomycin Technical Support support SABiosciences com www SABiosciences com 29 10 After 24 hours of transfection treat the cells as described in Table 12 with 1x 10x and 100x amount interfering peptide recombinant protein growth factor 1x is an smallest appropriate amount of small molecule or organic compound expected to modulate signaling pathway 11 To study the effect of interfering peptide recombinant protein growth factor we recommend harvesting cells 6 hours or 18 hours after treatment to develop luciferase assay 12 The luciferase assay can be developed by using Dual Luciferase Reporter Assay System from Promega Cat No 1910 Follow the manufacturer s protocol for developing the assay Expression of the Monster GFP reporter can be monitored via FACS flow cytometry fluorescent microscopy or standard fluorometry The spectral properties of the Monster Green Fluorescent Protein are sligh
33. rene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 25 ul of diluted SureFECT into each of the five tubes containing 25 ul of diluted constructs 1 1 ratio as detailed in Table 3 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in a culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2x10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately measure the cell density with a hemacytometer or automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliquot 50 ul of specific constructs snRNA SureFECT complexes into the appropriate wells 7 Add 100 ul of prepared cell suspension 210 cells ml in Opti MEM containing 5 of fetal bovine serum to each well containing construct snRNA SureFECT complexes This gives a final volume of 150 ul Mix gently by rocking the plate back and forth 8 Incubate cells at 37 C in a 5 COz2 incubator for 16 24 hours 9 After 16 24 hours of transfectio
34. riables to consider when optimizing the transfection conditions include cell density cell viability amount of DNA ratio of DNA to transfection reagent transfection complex formation time and transfection incubation time see the detailed protocols for our recommendations The positive control construct included with each Cignal Reporter Assay can be used for determining the optimal transfection conditions Optimization of assay condition The response rate in the Cignal Reporter Assay depends on the assay conditions conditions of the experimental treatment To obtain maximum response given by any stimulus perform dosing and time course studies The optimal amount of stimulus and the time of treatment must be obtained empirically for each experiment see different protocols for our recommendations Important recommendations for best results A Perform all transfections in triplicate to minimize variability among treatment groups B Include positive and negative controls in each experiment to obtain reliable results C Use low passage cells that are actively growing and are greater than 90 viable for maximal transfection efficiencies D Do not add antibiotics to media during transfection as this may cause cell death E Take care to always seed the same number of cells in each well in order to maximize the reproducibility of your experiment Technical Support support SABiosciences com www SABiosciences com 13 F Se
35. rum induces various signaling pathways leading to cross talk and high background Therefore use reduced amounts of serum 0 5 in the assay medium during the experimental treatment to minimize these serum effects B Generalized Transfection Protocols We recommend using reverse transfection protocols with the SureFECT transfection reagent throughout the Cignal Reporter Assays User Manual This is due to the time savings and improved reproducibility of using this method compared to traditional forward transfection methods However Cignal Reporter Assays also work well with traditional forward transfection methods and transfection reagents from other vendors Below are general protocol overviews for the Cignal Reporter Assays using either reverse or forward transfection approaches 1 Reverse Transfection Protocol Overview 1 DAY PROCEDURE Reverse Transfection 96 well Cell Culture Plate LEE Add SureFECT Transfection Reagent Cignal Reporter and Test Nucleic Acids 900 Seed Cells for Reverse Transfection DJQ DAY 1 LEE DAY 1 Prepare nucleic acid mixtures in appropriate ratios This may include any of the following combinations depending upon the experimental design we recommend carrying out each transfection condition in triplicate Experimental transfection i Cignal Reporter test nucleic acid expression plasmids shRNA plasmids or siRNAs Control transfections ii Cignal Reporter negative co
36. ry for visual confirmation of transfection It is also useful for transfection optimization studies Technical Support 888 503 3187 US 301 682 9200 Version 1 6 Tandem repeats TATA box A of TREs y TATA box j Hegre _ __ C CMV immediate early 7 enhancer promoter Figure 3 Schematic representation of constructs involved in Cignal GFP Reporter Assay A The inducible transcription factor responsive reporter expressing GFP B The GFP reporter controlled by a minimal promoter negative control C The constitutively expressing GFP construct positive control IMPORTANT NOTE There are a few reports in the literature of the CMV regulatory element being activated by certain stimuli see below We recommend that you confirm that the stimulus used in each Cignal reporter assay does not induce the CMV regulatory element in order to confirm that the CMV Renilla construct is the appropriate normalization construct for your experiment This can be done empirically by testing the impact of your stimulus on the Cignal positive control reporters which are each under the control of the CMV enhancer promoter cassette If your stimulus is one of the very few reported activators of the CMV regulatory element we advise using an alternative reporter as an internal control e W Bruening B Giasson W Mushynski and H D Durham 1998 Nucleic Acids Research 26 2 486 489 Activation of stress activated MAP protein kinases up regulates expres
37. sate for pipettor error when setting up transfection cocktails steps 1 through 4 Add 25 ul of Opti MEM to each of 5 polystyrene tubes avoid using DMEM along with the following Experimental transfection 1 ul 100 ng Cignal reporter 2 pmol sequence specific siRNA Control transfections 1 ul 100 ng Cignal reporter 2 pmol negative control siRNA 1 ul 100 ng Cignal negative control 2 pmol sequence specific siRNA 1 ul 100 ng Cignal negative control 2 pmol negative control siRNA 1 ul 100 ng Cignal positive control Mix each transfection cocktail gently Technical Support 888 503 3187 US 301 682 9200 Version 1 6 2 Prepare a SureFECT dilution for 5 tubes mentioned in step 1 by dispensing 1 5 ul of SureFECT into 125 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 25 ul of diluted SureFECT into each of the five tubes containing 25 ul of the diluted nucleic acids 1 1 ratio as detailed in Table 2 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in a culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 CO2
38. siRNAs Control transfections ii Cignal Reporter negative control for test nucleic acid iii Cignal Negative Control test nucleic acid expression plasmids shRNA plasmids or siRNAs iv Cignal Negative Control negative control for test nucleic acid v Cignal Positive Control Dilute transfection reagent into appropriate medium If you are using a transfection reagent other than SureFECT follow their manufacturer s protocol for transfection Add diluted transfection reagent to nucleic acid mixtures incubate at room temperature for 20 minutes Aliquot transfection complexes into wells containing overnight cell cultures Technical Support support SABiosciences com www SABiosciences com 15 C Co transfection Protocol for siRNA Reporter Assay The following protocol is designed to reverse transfect adherent cell line HEK 293H using SureFECT Transfection Reagent Cat No SA 01 in a 96 well plate format The Cignal Reporter Assay works well with transfection reagents from other vendors f you are using a transfection reagent other than SureFECT follow their manufacturer s protocol for optimizing transfection The Cignal Reporter Assay also works well using traditional forward transfection protocols Moreover if you are using plates or wells of different size adjust the components in proportion to the surface area see section IV H This is just a general quideline the optimal conditions amounts should be optimized accord
39. sion of transgenes driven by the cytomegalovirus immediate early promoter e Madhu S Malo Moushumi Mozumder Alexander Chen Golam Mostafa Xiao Bo Zhang Richard A Hodin 2006 Analytical Biochemistry 350 307 309 pFRL7 An ideal vector for eukaryotic promoter analysis Technical Support support SABiosciences com www SABiosciences com 11 Additional Materials Required Mammalian cell line cultured in the appropriate growth medium Cell culture medium and standard cell culture supplies 96 well tissue culture plates Multi channel pipettor and pipettor reservoirs Transfection reagent We recommend SureFECT SABiosciences Cat No SA 01 however other transfection reagents work equally well Polystyrene test tubes BD FALCON Cat 352099 Opti MEM Reduced Serum Medium Invitrogen Cat No 31985 062 Fetal bovine serum FBS Non essential amino acids NEAA Invitrogen Cat No 11140 050 Penicillin Streptomycin Hemacytometer Dual Luciferase Assay System o Dual Luciferase Reporter Assay System Promega Cat No E1910 This system requires cell lysis and is well suited for the rapid quantitation of both luciferase reporters when using luminometers with reagent auto injectors o Dual Glo Luciferase Assay System Promega Cat No E2920 This system is used to assay for both luciferase reporters on intact cells in growth medium This system can be used with any luminometer including those without reagent auto
40. tly red shifted compared to other commercially available GFP reporters We recommend using the standard FACS settings of an argon laser 488nm excitation and filters of 530 15 nm 530 30nm for emission When analyzing GFP expression via fluorescent microscopy or standard fluorometry we recommend using standard fluoroisothiocyanate FITC filters excitation of 470 20nm and an emission filter of 515nm long pass We found some components in DMEM interfere with SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that had been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support 888 503 3187 US 301 682 9200 Version 1 6 H Scaling up transfection experiments To transfect cells in different tissue culture formats vary the amounts of constructs number of cells and volume of SureFECT and medium used in proportion to the surface area aS shown in the Table 7 The parameters shown in Table 7 are standardized for HEK 293H cells Use these parameters as a starting point to optimize transfections for your cell line of int
41. total components needed on a per well basis for each condition to be tested Note that individual components must be added sequentially as instructed in the protocol 53s l e52 oss EEM fog lee o_ bo Shoes 5 oge Oss 8258 Sgsb8 B S58 OF v FB 352 HES Ww W z 1 a a 100 ng ne 25 ul 0 3 ul 25 ul 2 i a 200 ng 25 ul 0 3 ul 25 ul 3 i D 100 ng pa 25 ul 0 3 ul 25ul 32h 48h 4 hs i 200 ng 25 ul 0 3 ul 25 ul 5 i i is 100 ng pn 25 ul 0 3 pl 25 ul 6 ae aD 200 ng 25 ul 0 3 ul 25 ul 7 a is 100 ng Pe 25 ul 0 3 ul 25 ul 8 ne i 200 ng 25 ul 0 3 pl 25 ul 9 a a 25 ul 0 3 ul 25 ul Carrier DNA means any empty plasmid such as a pUC or a pBR plasmid 1 The recommended experimental setup on a per well basis follows Please note that we recommend setting up multiple replicates for each condition and preparing transfection cocktail volumes sufficient for transfecting multiple wells In addition we advise always taking 5 10 extra amounts of nucleic acid Opti MEM serum free culture medium and SureFECT to compensate for pipettor error when setting up transfection cocktails steps 1 through 4 Technical Support 888 503 3187 US 301 682 9200 Version 1 6 Add 25 ul of Opti MEM to each of 9 polystyrene tubes avoid using DMEM along with the following Experimental transfections 1 ul 100 ng Cignal reporter 100 ng experimental vector expressing gene of i
42. try fluorescent microscopy or standard fluorometry The spectral properties of the Monster Green Fluorescent Protein are slightly red shifted compared to other commercially available GFP reporters We recommend using the standard FACS settings of an argon laser 488nm excitation and filters of 530 15 nm 530 30nm for emission When analyzing GFP expression via fluorescent microscopy or standard fluorometry we recommend using standard fluoroisothiocyanate FITC filters excitation of 470 20nm and an emission filter of 515nm long pass Technical Support support SABiosciences com www SABiosciences com 17 We have found that some components in DMEM interfere with SureFECT transfection protocol However DMEM has no effect on the performance of Cignal Reporter Assays Cells that have been passed 1 3 or 1 4 the day before are generally more easily transfected than cells that have reached a confluent state at the time of use In most cases cells grow well in Opti MEM serum reduced growth medium with 3 5 FBS due to extra growth factors and nutrients supplied in Opti MEM Cell should reach 50 90 confluence once attached to the wells otherwise increase the cell numbers Technical Support 888 503 3187 US 301 682 9200 Version 1 6 D Co transfection Protocol for shRNA Reporter Assay The following protocol is designed to reverse transfect adherent cell line HEK 293H using SureFECT Transfection Reagent Cat No
43. ve control Mix each transfection cocktail gently 2 Prepare a SureFECT dilution for 9 tubes mentioned in step 1 by dispensing 2 7 ul of SureFECT into 225 ul of Opti MEM serum free culture medium for every well dilute 0 3 ul of SureFECT in 25 ul of Opti MEM serum free culture medium in a polystyrene test tube Mix gently and set the tube at room temperature for 5 minutes 3 After the 5 minute incubation add 100 ul of diluted SureFECT to the two tubes mentioned in step 1 containing equal volume 100 ul of diluted Cignal reporter and add 25 ul of diluted SureFECT into the positive control tube containing 25 ul of diluted constructs 1 1 ratio as detailed in Table 6 4 Mix gently and incubate for 20 minutes at room temperature to allow complex formation to occur 5 Meanwhile wash cells in culture dish once with Dulbecco s PBS without calcium and magnesium and treat with 1 3 ml trypsin EDTA for 2 5 minutes at 37 C in a humidified atmosphere containing 5 CO2 Suspend the cells in 7 9 ml of Opti MEM containing 5 of fetal bovine serum then centrifuge the cells down remove the supernatant and resuspend the cells to 2x10 cells ml in Opti MEM containing 5 of fetal bovine serum and 1 NEAA To ensure reproducible transfection results it is important to accurately determine the cell density with a hemacytometer or an automated cytometry device 6 After the 20 minute incubation for complex formation is completed aliqu
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