NeuroPORTER ® Transfection Reagent
Contents
1. Product Name Cat No Quantity GenePORTER 2 Transfection Reagent T202007 75 reactions 0 75 ml GenePORTER 2 Transfection Reagent T202015 150 reactions 1 5 ml GenePORTER 2 Transfection Reagent 1202075 750 reactions 5 x 1 5 ml Product Support Telephone 858 457 1919 Fax 858 623 9494 OR 888 428 0558 US toll free E mail tech genlantis com Web http Awww genlantis com For a complete list of international distributors visit our web site at www genlantis com Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 858 457 1919 or 888 428 0558 US Toll Free Introduction NeuroPORTER Transfection Reagent is a novel cationic lipid specially formulated for optimal transfection in neuronal cells including primary neurons differentiated post mitotic neurons neuronal cell lines and glial cells NeuroPORTER Transfection Reagent is much easier to use than the traditional viral delivery method for transfecting DNA into neuronal cells NeuroPORTER Transfection Reagent is compatible with serum eliminates the need to change media following transfection An included DNA Diluent is designed to facilitate DNA lipid complex lipoplex formation and enhance the transformation efficiency in certain neuronal cells such as NT2 not recommended for primary and differentiated neurons Compared to other commercially available transfection reagents NeuroPORTER provides sup2. Telephone 858 457 1919 Fax 858 623 9494 Email licensing genlantis com Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 858 457 1919 or 888 428 0558 US Toll Free TABLE OF CONTENTS Page OVERVIEW Purchaser Notification 4 se secs sexi ceayeis cecettestessgnneseeatanheengeng eecautensetageaatecs E EE ETEEN 3 Keb COmte ints ise fete a aie aea eee leas dada scence haar taa be a Save ge a Sok custo tan on SS 5 Shipping and Stoja genein ini baad bases fasta dean a bs ee dot A EE aE INE ode A aa Ns 5 Related Prod cts sas heee nie r a EEE a E E eE a duke whe ear dees ee Ree 5 Product Support se a a a a Lace eae a Lane a 5 TAT OCU CHOI asss eran eee oA ra ET RAA ES na AR AA E O aa a RAA SE 6 METHODS AND PROCEDURES Transfection of Primary Rat Hippocampal Neurons sssssssssessesseesssssesersessssesesseseesesrssesresessss 6 Transfection of Other Primary Neurons ccccccescessceseceseceeeeeeeeesseesseeeseecseceseenseeeeeseeeeneeesss 8 Transfection of Neuronal Cell Lines 20 0 eccescceseeeeeecesecseeeeaecaeeeecesecaecaaeeneeseceaeeaeeeeenaeeas 10 Transfection of Differentiated Post Mitotic Neurons and Glial Cell Lines cee 12 APPENDIX Q ality Control remisie ee cecd selected deze e ede had oases eh ines od dentate tate dad ee a 14 Examples of Optimization of Transfection Conditions cecceseseesceseceeeeeeeeceeeeaeeeeeeaeeaeees 14 Version MV081105 Genlantis a Division of Gene
3. 2 ug 50 ul 3 ug 75 ul Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 858 457 1919 or 888 428 0558 US Toll Free 15 3 Optimization conditions for differentiated post mitotic neurons and glial cell line transfection in 24 well plates Follow the general protocol to prepare the DNA NeuroPORTER complexes We do not recommend using the DNA Diluent for differentiated post mitotic neurons and glial cells Condition DNA dilutions in NeuroPORTER dilutions in serum Total Final DNA serum free medium free medium Volume Concentration 10 ug in 250 ul 50 ul in 200 ul Vt 250 pl 500 ul 20 ug ml 75 ulin 175 ul Vt 250 ul 100 ul in 150 ul Vt 250 pl i 125 ul in 125 pl Vt 250 ul i X E 150 pl in 100 ul Vt 250 pl n DW nN BI WwW NM 200 ul in 50 ul Vt 250 pl i Add the appropriate volume of complexes solution directly to your cells as illustrated below Volume of DNA NP Complexes CONDITIONS Transferred DNA well well 0 5 ug 25 ul 4 a Lug sop M K 2 ug 100 O ie 3 ug 150 ul Te Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 858 457 1919 or 888 428 0558 US Toll Free
4. Cell Lines 3 1 3 2 3 3 3 4 Hydrate NeuroPORTER lipid film at room temperature with 1 5 ml of the hydration buffer Vortex for 30 60 seconds at top speed Store the hydrated reagent at 4 C and vortex briefly before use Dilute the hydrated NeuroPORTER reagent with serum free medium Refer to Table 6 for the appropriate volume of serum free medium Table 6 Volumes of Transfection Reagents DNA Diluent NeuroPORTER Serum Free Medium ul for NeuroPORTER Dilute the DNA with the DNA Diluent and incubate 1 to 5 minutes at room temperature Refer to Table 6 for the appropriate volume of DNA Diluent Do not incubate DNA with the DNA Diluent for longer than 5 minutes Avoid vortexing the DNA diluent Although NeuroPORTER consistently delivers high transfection efficiencies in order to obtain maximum efficiency in particular cell types some optimization may be needed The two critical variables are the ratio of NeuroPORTER reagent to DNA and the quantity of DNA used For optimization first maintain a fixed ratio of NeuroPORTER reagent to DNA and then vary the DNA quantity over the suggested range If necessary optimize the ratio of NeuroPORTER reagent to DNA by using 1 25 to 12 5 ul of reagent for each 1 ug of DNA Use a low DNA quantity to optimize this ratio Following this process cell number can also be optimized See the Appendix for examples Add the DNA solution to the diluted NeuroPORTER Transfection Reagent Incubate
5. at room temperature for 5 to 10 minutes to allow the NeuroPORTER DNA complexes to form Do not incubate the DNA solution with the NeuroPORTER Transfection Reagent for longer than 30 minutes Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 10 858 457 1919 or 888 428 0558 US Toll Free 3 5 Add your complexes directly to the cells growing in serum containing culture medium Refer to Table 7 for suggested cell numbers for specific tissue culture dishes Refer to Table 8 for appropriate medium volumes Cells plated the day before transfection should be 50 to 70 confluent on the day of transfection Table 7 Suggested Cell Culture Conditions Table 8 Medium Volumes and DNA Amount for Transfection of Neuronal Cell Lines for Various Culture Dishes Tissue Number of Cells Well Tissue DNA Medium Culture Dish Culture Dish ug Volume ml 25 30 x 10 125 150 x 10 250 300 x 10 500 600 x 10 1 1 5 x 10 2 5 3 x 10 3 6 Add fresh growth media as needed 24 hours post transfection Depending on the cell type and promoter activity the assay for the reporter gene can be performed 24 to72 hours following transfection For some cell types the old media can be replaced with fresh media at this step The same protocol can be used to produce stably transfected cells 48 to 72 hours post transfection put the cells in fresh medium containing the appropriate selection antibiotic It is important to wait at
6. least 48 hours before exposing the transfected cells to the selection media For some cell types it may be necessary to wait as long as 4 to 5 days before applying the selection condition Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 11 858 457 1919 or 888 428 0558 US Toll Free 4 Transfection of Differentiated Post Mitotic Neurons and Glial Cell Lines 4 1 Hydrate NeuroPORTER lipid film at room temperature with 1 5 ml of the hydration buffer Vortex for 30 60 seconds at top speed Store the hydrated reagent at 4 C and vortex briefly before use 4 2 Dilute the hydrated NeuroPORTER reagent with serum free medium Refer to Table 9 for the appropriate volume of serum free medium Table 9 Volumes of Transfection Reagents DNA Serum Free Medium NeuroPORTER Serum Free Medium ug for DNA ul for NeuroPORTER 4 3 Dilute the DNA with the serum free medium Refer to Table 9 for the appropriate volume of serum free medium Although NeuroPORTER consistently delivers high transfection efficiencies in order to obtain maximum efficiency in particular cell types some optimization may be needed The two critical variables are the ratio of NeuroPORTER reagent to DNA and the quantity of DNA used For optimization first maintain a fixed ratio of NeuroPORTER reagent to DNA and then vary the DNA quantity over the suggested range If necessary optimize the ratio of NeuroPORTER reagent to DNA by using 5 to 20 ul of reagent
7. some optimization may be needed The two critical variables are the ratio of NeuroPORTER reagent to DNA and the quantity of DNA used For optimization of the DNA quantity used maintain a fixed ratio of NeuroPORTER reagent to DNA and then vary the DNA quantity over a suggested range see Table 5 See the Appendix for examples Add the DNA solution to the diluted NeuroPORTER Transfection Reagent Incubate at room temperature for 5 to 10 minutes to allow the NeuroPORTER DNA complexes to form Do not incubate the DNA solution with the NeuroPORTER Transfection Reagent for longer than 30 minutes Add your complexes directly to the cells growing in serum containing culture medium Refer to Table 5 for suggested medium volumes Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 858 457 1919 or 888 428 0558 US Toll Free Table 5 Medium Volumes and DNA Amount for Various Culture Dishes Tissue DNA Medium Culture Dish ug Volume ml 96 well 24 well 12 well 6 well 60 mm 100 mm 2 6 Add fresh growth media as needed 24 hours post transfection Depending on the cell type and promoter activity the assay for the reporter gene can be performed 24 to72 hours following transfection For some cell types the old media can be replaced with fresh media at this step Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 9 858 457 1919 or 888 428 0558 US Toll Free 3 Transfection of Neuronal
8. Medium containing 2X concentration of B27 onto the cells Version MV081105 Perform assay for gene expression after 24 48 hours Genlantis a Division of Gene Therapy Systems Inc 858 457 1919 or 888 428 0558 US Toll Free 2 Transfection of Other Primary Neurons 2 1 2 2 2 3 2 4 2 5 Hydrate the NeuroPORTER lipid vial at room temperature with 1 5 ml of the hydration buffer Vortex for 30 60 seconds at top speed Store the hydrated reagent at 4 C and vortex briefly before use Dilute the hydrated NeuroPORTER reagent with serum free medium Refer to Table 4 for the appropriate volume of serum free medium Table 4 Volumes of Transfection Reagents DNA Serum Free Medium NeuroPORTER Serum Free Medium ug for DNA ul for NeuroPORTER uD Although NeuroPORTER has been optimized for specific cell culture conditions optimization may be needed to achieve maximum transfection efficiency The two critical variables are the ratio of NeuroPORTER reagent to DNA and the quantity of DNA used For optimization of the ratio of NeuroPORTER reagent to DNA start by using 2 5 to 15 ul of reagent for each I ug of DNA Use a fixed amount of DNA or vary the amount as suggested in the Appendix to optimize this ratio Dilute the DNA with the serum free medium do not use the DNA Diluent for primary neurons Refer to Table 4 for the appropriate volume of serum free medium To obtain maximum efficiency in particular cells
9. NeuroPORTER Transfection Reagent Instruction Manual Catalog Number T400150 T400750 Z Genlantis Genlantis A Division of Gene Therapy Systems Inc 10190 Telesis Court San Diego CA 92121 USA Telephone 858 457 1919 US Toll free 888 428 0558 Fax 858 623 9494 E mail techl genetherapysystems com Web Site http www genetherapysystems com PAGE INTENTIONALLY LEFT BLANK Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 858 457 1919 or 888 428 0558 US Toll Free Purchaser Notification Limited License The purchase price paid for the NeuroPORTER Transfection Reagent kit by end users grants them a non transferable non exclusive license to use the kit and or its separate and included components as listed in the Kit Contents section This kit is intended for internal research only by the purchaser Such use is limited to the transfection of nucleic acid into neuronal cells as described in the product manual Furthermore research only use means that this kit and all of its contents are excluded without limitation from resale repackaging or use for the making or selling of any commercial product or service without the written approval of Genlantis a division of Gene Therapy Systems Inc Genlantis Separate licenses are available from Genlantis for the express purpose of non research use or applications of the NeuroPORTER Transfection Reagent To inquire about such lice
10. Therapy Systems Inc 858 457 1919 or 888 428 0558 US Toll Free OVERVIEW Kit Contents Each NeuroPORTER Transfection Reagent kit Cat No T400150 contains sufficient material for 75 to 300 transfection reactions depending on the cell type Each NeuroPORTER Transfection Reagent kit Cat No T400750 contains sufficient material for 375 to 1500 transfection reactions depending on the cell type Each reaction is for transfecting 2 ug of DNA Item Description Quantity NeuroPORTER Dried NeuroPORTER lipid film 1 vial Cat T400150 Transfection Reagent 5 vials Cat T400750 Hydration Buffer Transfection grade hydration buffer used to 1 5 ml Cat T400150 hydrate NeuroPORTER dried lipid film 5 x 1 5 ml Cat T400750 before transfection DNA Diluent Solution for diluting DNA for optimal 7 5 ml Cat T400150 transfection efficiency in neuronal cell lines 5 x 7 5 ml Cat T400750 Shipping and Storage The NeuroPORTER Transfection Reagent kit is shipped at room temperature For maximum stability store all reagents at 4 C upon receipt All components are stable for at least one year if stored properly Related Products hCMV Mammalian Expression Vectors Product Name Cat No Quantity phCMV1 Vector Kit P003100 25 ug phCMV 2 Vector Kit P003200 25 ug phCMV3 Vector Kit P003300 25 ug For efficient transfection and high level expression in non neuronal cells
11. erior transfection efficiency and minimized cytotoxicity Cell type specific protocols are developed for NeuroPORTER Transfection Reagents to ensure optimal transfection results METHODS AND PROCEDURES 1 Transfection of Primary Rat Hippocampal Neurons 1 1 Seed primary rat hippocampal cells in poly D lysine coated plates Becton Dickinson Labware in the numbers listed in Table 1 below using the following Plating Medium Neurobasal medium Invitrogen Cat No 21103 049 supplemented with B27 0 5 mM L glutamine and 25 uM glutamate Incubate the cells at 37 C in 5 CO for 72 hours Table 1 Suggested Cell Plating Numbers Tissue Culture Plate Cell Number Plating Medium Volume 96 well 15 000 cells well 0 125 ml 24 well 100 000 cells well 0 5 ml 12 well 200 000 cells well 1 0 ml 6 well 500 000 cells well 2 0 ml 1 2 After 72 hours of incubation remove 2 volume of the Plating Medium and replace with the following Culture Medium Neurobasal medium supplemented B27 and 0 5 mM L glutamine no 25 uM glutamate Continue incubation for an additional 24 hours 1 3 Hydrate the NeuroPORTER lipid vial at room temperature with 1 5 ml of the hydration buffer Vortex for 30 60 seconds at top speed Store the hydrated reagent at 4 C and vortex briefly before use 1 4 Dilute the DNA and hydrated NeuroPORTER reagent with serum free medium do not use the DNA Diluent for primary neurons Refer to Tables 2 and 3 for
12. for each 1 ug of DNA Use a low DNA quantity to optimize this ratio Following this process cell numbers can also be optimized See the Appendix for examples 4 4 Add the DNA solution to the diluted NeuroPORTER Transfection Reagent Incubate at room temperature for 5 to 10 minutes to allow the NeuroPORTER DNA complexes to form Do not incubate the DNA solution with the NeuroPORTER Transfection Reagent for longer than 30 minutes 4 5 Add your complexes directly to the cells growing in serum containing culture medium Refer to Table 10 for suggested cell number according to culture dishes size and cell types Refer to Table 11 for appropriate medium volumes Cells plated the day before transfection should be 50 to 70 confluent on the day of transfection Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 12 858 457 1919 or 888 428 0558 US Toll Free Table 10 Suggested Cell Culture Conditions for Table 11 Medium Volumes and DNA Transfection of Differentiated Neurons and Glial Cells Amount for Various Culture Dishes Tissue Cells Well Cells Well Tissue DNA Medium Culture Dish Diff Neurons Glial Cells Culture Dish ug Volume ml 96 well 96 well 24 well 24 well 12 well 12 well 6 well 6 well 60 mm 60 mm 100 mm 100 mm 4 6 24 hours post transfection add fresh growth media as needed Depending on the cell type and promoter activity the assay for the reporter gene can be performed 24 to72 hours follow
13. ing transfection For some cell types the old media can be replaced with fresh media at this step Also the same protocol can be used to produce stably transfected cells 48 to 72 hours post transfection put the cells in fresh medium containing the appropriate selection antibiotic It is important to wait at least 48 hours before exposing the transfected cells to the selection media For some cell types it may be necessary to wait as long as 4 to 5 days before applying the selection condition Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 13 858 457 1919 or 888 428 0558 US Toll Free APPENDIX Quality Control To assure the performance of each lot of the NeuroPORTER reagent we pre qualify the chemical synthesis of NeuroPORTER lipid by mass spectrometry and thin layer chromatography The final product is further tested by in vitro B galactosidase transfection assay in NT2 neuronal precursor cell Each lot shall have an acceptance specification of gt 70 of the activity of the Reference lot Examples of Optimization of Transfection Conditions 1 Optimization conditions for primary neuron transfection in 24 well plates Follow the general protocol to prepare the DNA NeuroPORTER complexes We do not recommend using the DNA Diluent for primary neurons Condition DNA dilutions in NeuroPORTER dilutions in serum Total Final DNA serum free medium free medium Volume Concentration 10 ug in 250 ul 25 ul in 225 ul V
14. nses or to obtain permission to transfer or use the enclosed material contact the Director of Licensing at Genlantis Purchasers may terminate this License at any time by returning all NeuroPORTER Transfection Reagent material and documentation to Genlantis or by destroying all NeuroPORTER Transfection Reagent kit components Purchasers are advised to contact Genlantis with the notification that a NeuroPORTER Transfection Reagent kit is being returned in order to obtain a refund and or to expressly terminate a research only license granted through the purchase of the kit s This document covers in full the terms of the NeuroPORTER Transfection Reagent research only license and does not grant any other express or implied license The laws of the State of California shall govern the interpretation and enforcement of the terms of this License Product Use Limitations The NeuroPORTER Transfection Reagent and all of its components are developed designed intended and sold for research use only They are not to be used for human diagnostic or included used in any drug intended for human use All care and attention should be exercised in the handling of the kit components by following appropriate research lab practices For more information or for any comments on the terms and conditions of this License please contact Director of Licensing Genlantis a Division of Gene Therapy Systems Inc 10190 Telesis Court San Diego CA 92121
15. recommended DNA NeuroPORTER and serum free medium volumes for different tissue culture plates Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 858 457 1919 or 888 428 0558 US Toll Free Table 2 Volumes of Transfection Reagents DNA Serum Free NeuroPORTER Serum Free ug Medium for DNA ul Medium For m NeuroPORTER ul 0 1 0 5 12 5 2 5 10 1 3 25 5 20 2 4 37 5 7 5 30 4 6 62 5 12 5 50 Add the DNA solution to the diluted NeuroPORTER Transfection Reagent Mix by pipetting up and down several times Incubate at room temperature for 10 minutes to allow the NeuroPORTER DNA complexes to form Do not incubate for longer than 30 minutes Table 3 Medium Volumes and DNA Amount for Various Culture Dishes Tissue Culture DNA Serum Free Medium Total Transfection Dish ug Volume ml Volume ml 96 well 0 1 0 5 0 1 0 125 24 well 1 3 0 45 0 5 12 well 2 4 0 925 1 0 6 well 4 6 1 375 1 5 1 6 Remove the Plating Medium from the cells and add the volume of serum free medium indicated in Table 3 to each well 1 7 Apply the DNA NeuroPORTER complexes from step 1 5 to each well The total transfection volume at this step is indicated in Table 3 1 8 Gently mix the DNA NeuroPORTER serum free medium by swirling and place the cells in a 37 C incubator with 5 COs 1 9 After two hours of incubation add one additional volume of fresh Culture
16. t 250 ul 500 ul 20 ug ml 50 ul in 200 pl Vt 250 pl 4 75 ulin 175 pl Vt 250 pl 100 pl in 150 ul Vt 250 ul i 125 ul in 125 ul Vt 250 ul 150 ul in 100 ul Vt 250 ul Alal Bl WwW NR Add the appropriate volume of complexes solution directly to your cells as illustrated below Volume of DNA NP Complexes CONDITIONS Transferred DNA well well 0 5 ug au M R 1 pg sou M K 2 ug 100 RO 3 pg 150 ul amp Version MV081105 Genlantis a Division of Gene Therapy Systems Inc 14 858 457 1919 or 888 428 0558 US Toll Free 2 Optimization conditions for neuronal cell line transfection in 24 well plates Follow the general protocol to prepare the DNA NeuroPORTER complexes We recommend using the DNA Diluent for neuronal cell lines such as NT2 Condition DNA Diluent NeuroPORTER dilutions in serum free medium Total Volume Final DNA Concentration 10 pg in 125 ul 12 5 ulin 112 5 pl Vt 125 pl 250 ul 40 ug ml 25 ul in 100 pl Vt 125 pl 50 ul in 75 ul Vt 125 ul 75 ul in 50 ul Vt 125 ul 100 ul in 25 ul Vt 125 ul Din AJ WL NM rR 125 ul NeuroPORTER Add the appropriate volume of complexes solution directly to your cells as illustrated below Volume of DNA NP Complexes CONDITIONS Transferred DNA well well 1 2 3 4 5 6 0 5 ug 12 5 ul 4 1 ug 25 ul
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