Magnetofection
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1. cell type or cell line is not listed below this does not imply that Magnetofection is not going to work OZ Biosciences is going to maintain an updated list of cells successfully tested that is available on the website www ozbiosciences com For the cells listed some reagents have not been tested so far as indicated by n d not determined Standard Transfection Magnetofection Primary Cells Confluent Primary Human Keratinocytes Transfected with a commercial reagent L or PolyMag Reporter Gene GFP Primary Human Keratinocytes Transfected with a commercial reagent F CombiMag Reporter Gene GFP Primary Chondrocytes Pig Transfected with a commercial reagent F CombiMag Reporter Gene Bgalactosidase Cell Lines CT 26 Colon Carcinoma Mouse Transfected with a commercial reagent D or PolyMag Reporter Gene GFP HUVECC Primary Human Transfected with a commercial reagent E CombiMag Fluorescent Oligonucleotides We are grateful to the laboratories of Dr C Plank Technical University Munich and of Dr F Kroetz Ludwig Maximilians University Munich for kindly providing these data OZ Biosciences Protocol Magnetofection www ozbiosciences com 4 Cell Line Cell Type Source PolyMag CombiMag 293 T EBNA Set baoetie aa a Fares tons Fama Vo a a a HUVEC Endothelial Cells primary Human 58 Priam a a a a a e E Ew a e a 2 2 Primary Airway2. 1 Scherer F Anton M Schillinger U Henke J Bergemann C Kruger A Gansbacher B and Plank C Magnetofection enhancing and targeting gene delivery by magnetic force in vitro and in vivo Gene Ther 2002 Jan 9 2 102 9 2 Krotz F Wit C Sohn HY Zahler S Gloe T Pohl U and Plank C Magnetofection A highly efficient tool for antisense oligonucleotide delivery in vitro and in vivo Mol Ther 2003 May 7 5 700 10 3 Plank C Schillinger U Scherer F Bergemann C Remy JS Krotz F Anton M Lausier J and Rosenecker J The magnetofection method using magnetic force to enhance gene delivery Biol Chem 2003 May 384 5 737 47 4 Plank C Anton M Rudolph C Rosenecker J and Krotz F Enhancing and targeting nucleic acid delivery by magnetic force Expert Opin Biol Ther 2003 Aug 3 5 745 58 6 Related Products Description Reference B Galactosidase assay kit with ONPG GO 10001 B Galactosidase assay kit with CPRG GC 10002 X Gal staining kit GX 10003 DreamFect 0 5mL DF 40500 FlyFectin 0 5mL FF 50500 GeneBlaster SelectionKit GB 20010 Please feel free to contact us for all complementary information and remember to visit our website to stay informed on the latest breakthrough technologies and updated on our complete product list OZ Biosciences Protocol Magnetofection www ozbiosciences com 14 Purchaser Notification Limited License The purchase of the Magnetofection Reagent grants the purch
3. 1 2x10 2 4 1 mL 6 well 2 4x 10 2 6 2 mL 60 mm dish 5 10x10 6 8 5 mL 90 100 mm dish 10 2010 8 12 10 mL T 75 flask 20 50 107 15 25 15 20 mL OZ Biosciences Protocol Magnetofection www ozbiosciences com 6 The same protocol can be used to produce stably transduced cells except that 48 hours post transfection cells are transferred to fresh medium containing the appropriate antibiotics for selection It is important to wait at least 48 hours before exposing the transduced cells to selection media Vectors are prepared in medium without serum and supplement or in physiological saline because serum may interfere with vector assembly According to the standard Magnetofection protocol the serum and supplement free vector cocktail is added to the cells that are covered with complete medium Therefore the addition of the transfection cocktail will result in the dilution of supplements such as serum antibiotics or other additives of your standard culture medium Although a medium changes after Magnetofection is not required for most cell types it may be necessary for cells that are sensitive to serum supplement concentration Alternatively the cells may be kept in serum free medium during Magnetofection In this case a medium change will be required after Magnetofection 4 3 PolyMag The protocol is as simple as follows Use 1 uL of PolyMag per ug of DNA 1 Before each use vortex the PolyMag material Add 1 to 10 uL of PolyM
4. ARTICLES Shipping condition Room Temperature 2 Applications 2 1 Nucleic Acids Dose Response and Transfection Kinetics Save Materials Save Times param agnetic vehicle plusm agnetic field param agnetic vehicle no magnetic field standard gene tansfer response expression lev response expression lev dose yg DNA process time min DNA dose response profile Transfection kinetics NIH 3T3 NIH 3T3 cells were transfected cells were incubated with a with a commercial transfection commercial transfection reagent G reagent L CombiMag with and CombiMag with and without without the magnetic field for 15 positioning on the magnetic plate min Luciferase expression was for the indicated time spans assayed after 24 hours Luciferase expression was assayed after 24 hours 2 2 Nucleic Acids Types and Vectors The CombiMag reagent can be combined with any nucleic acid any cationic polymer based and lipid based transfection reagent and also with virus adenovirus or retrovirus Nucleic Acid or Virus Type PolyMag CombiMag DNA plasmid Antisense Oligonucleotides siRNA Adenovirus Retrovirus OZ Biosciences Protocol Magnetofection www ozbiosciences com 3 2 3 Cell Types Magnetofection is generally applicable on numerous cell types This technology has been tested successfully on a variety of immortalized cell lines as well as primary cells see table below If a particular
5. Epithelium i Primary Aortic Endothelial Cells PAEC Human Ni Bovine Primary Carotid Artery Smooth Muscle Cells 1 L lt 4 a N 5 Lj a Lj a L 1 Q 2 S 2 Lj lt lt lt Lj lt lt lt a a Primary Fibrochondrocytes ey eee Th ee fees st Primary Keratinocytes Human n d q Mouse Primary Peripheral Blood Lymphocytes Human n d y 12 Mouse Successfully tested in combination with several commercially available transfection reagents F L D E and adenovirus For more information see the bibliographic references at the end of this manual OZ Biosciences Protocol Magnetofection www ozbiosciences com 5 3 Magnetofection Apparatus Apart from suitable magnetic nanoparticles Magnetofection requires appropriate magnetic fields A magnetic plate especially designed for Magnetofection is provided to exert these specific magnetic fields Its special geometry not only produces strong magnetic fields under each well of 96well plates but is also applicable for other plate formats T 75 flasks 60 amp 100 mm dishes 6 12 and 24 well plates In the larger plate formats the magnetic plate will produce a pattern of higher and lower densities of transfected cells according to the geometry of the magnetic field lines 4 Example Protocols 4 1 General Considerations The instructions given below represent sample p
6. Magnetofection 3 PRIMARY CELLS CELL LINES Magnetofection Instruction Manual Magnetofection is a novel simple and highly efficient in vitro and in vivo transfection method List of Magnetofection Kits Catalog Description Volume uL Size number of Number of Number transfection ug of transfections DNA 96 well plates PN 30100 PolyMag II 100 100 1000 PN 30200 PolyMag II 200 200 2000 PN 31000 PolyMag II 1000 1000 10000 CM 20100 CombiMag 100 100 1000 CM 20200 CombiMag 200 200 2000 CM 21000 CombiMag 1000 1000 10000 KM 30200 Magnetofection Selection Kit 200 2 X 100 200 2000 KC 30296 Magnetofection Starting Kit 200 2 X 100 200 2000 MF 10096 Magnetic Plate N A N A N A Contains 1 vial of each reagent PolyMag and CombiMag Contains 1 vial of each reagent PolyMag and CombiMag plus a Magnetic Plate Use the content of the table above to determine the appropriate catalog number for your needs You can order these products by contacting us For all other supplementary information do not hesitate to contact our dedicated technical support OZ BIOSCIENCES Parc Scientifique et Technologique de Luminy BP13 13273 Marseille Cedex 9 France Tel 33 0 4 91 82 81 72 Fax 33 0 4 91 82 81 70 E mail contact ozbiosciences com Site Internet www ozbiosciences com Patent Pending OZ BIOSCIENCES The art of delivery systems OZ Biosciences Protocol Magnet
7. The laws of the French Government shall govern the interpretation and enforcement of the terms of this License Product Use Limitations The Magnetofection 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 use of the kit components by following proper research laboratory practices For more information or for any comments on the terms and conditions of this License please contact Director of Business Development OZ Biosciences Parc Scientifique et Technologique de Luminy BP13 13273 Marseille Cedex 9 France Tel 33 0 4 91 82 81 74 Fax 33 0 4 91 82 81 70 E mail contact ozbiosciences com OZ Biosciences Protocol Magnetofection www ozbiosciences com 15
8. ag according to the DNA amount to a microtube or to a microwell U bottom well is preferred to get a better mixing If required and for doses less than 1 uL in your protocol predilute PolyMag with deionized water 2 Dilute 1 to 10 ug of DNA to 200 uL with serum and supplement free culture medium such as DMEM 3 Add the 200 uL DNA solution to the PolyMag solution and mix immediately by vigorous pipetting 4 After 20 to 30 minutes of incubation add the 200 uL of complexes to the cells The total transfection volumes per well culture medium PolyMag mixture are suggested in the table above Note to transfect cells in duplicate prepare your DNA PolyMag complexes as described previously and transfer 100 uL of the resulting mixture to each well containing the cells to be transfected 5 Place the cell culture plate upon the magnetic plate for 5 to 20 minutes 6 Remove the magnetic plate Optionally perform a medium change 7 Cultivate the cells under standard conditions until evaluation of transgene expression 4 4 CombiMag Until now a universal method allowing to enhance the efficiency of synthetic non viral and viral gene delivery systems was lacking Magnetofection is the only existing method answering these needs The conducted studies have shown that Magnetofection e Increases the efficiencies of commercial transfection reagents amp reduces the required DNA doses e Significantly improves the efficiencies of all t
9. agnetic plate 4 Continue to incubate for 15 minutes 5 Carefully remove the medium supernatant from the cells and replace with fresh complete medium while the culture plate remains positioned on the magnetic plate Be careful not to aspirate the magnetically sedimented cells 6 Remove culture plate from magnetic plate 7 Continue to cultivate cells as desired until evaluation of transgene expression OZ Biosciences Protocol Magnetofection www ozbiosciences com 10 5 Appendix 5 1 Protocol Optimization We strongly advise you to optimize your transfection and or infection conditions in order to get the best out of Magn tofection Several parameters can be optimized e Nucleic acid or viral vector dose used e Ratio of CombiMag PolyMag to nucleic acid virus o Cell type and cell density e Incubation time OZ Biosciences team has investigated numerous factors during the course of the R amp D program Based on our experience we recommend that you optimize one parameter at a time and start from the experimental procedures described above section 4 1 Start by optimizing the ratio PolyMag DNA or CombiMag transfection reagent or virus To this end use a fixed amount of DNA and transfection reagent Vary the amount of CombiMag PolyMag from 0 25 to 5uL ug of DNA The ratio PolyMag or CombiMag DNA can be changed by doubling or multiplying the volumes of the reagents used Similarly the reagents can be pr
10. aser a non transferable non exclusive license to use the kit and or its separate and included components as listed in section 1 Kit Contents This reagent is intended for in house research only by the buyer Such use is limited to the transfection of nucleic acids as described in the product manual In addition 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 OZ Biosciences Separate licenses are available from OZ Biosciences for the express purpose of non research use or applications of the Magnetofection Reagent To inquire about such licenses or to obtain authorization to transfer or use the enclosed material contact the Director of Business Development at OZ Biosciences Buyers may end this License at any time by returning all Magnetofection Reagent material and documentation to OZ Biosciences or by destroying all Magnetofection Reagent components Purchasers are advised to contact OZ Biosciences with the notification that a Magnetofection Reagent kit is being returned in order to be reimbursed and or to definitely terminate a license for internal research use only granted through the purchase of the kit s This document covers entirely the terms of the Magnetofection Reagent research only license and does not grant any other express or implied license
11. ediluted in deionized water and aliquots of the resulting dilutions are incubated with DNA or pre formed DNA complexes Finally the different components can be serially diluted to very low concentrations 2 Thereafter change the nucleic acid dose or viral MOI with a fixed ratio of PolyMag DNA or CombiMag transfection reagent or virus that has been previously optimized For this purpose you can perform a serial dilution of a preformed magnetic vector complex 3 After having identified the correct quantity of CombiMag PolyMag nucleic acid transfection reagent commercial or viral vector you could pursue the process by optimizing the cell number as well as the incubation times for the complex formation and for the magnetic field application 5 2 Protocol Optimization in a 96 well format Adherent cells For adherent cells seed the cells at the desired density in a 96 well plate the day prior or at least several hours prior transfection in a total of 150 uL medium per well 1 In four tubes dilute 7 2 ug of DNA or DNA transfection reagent complex each to 346 ul with serum and supplementfree medium e g DMEM 2 Provide 3 6 7 2 10 8 and 14 4 uL respectively of PolyMag in case of DNA or CombiMag in case of DNAttransfection reagent complex in well Al A4 A7 and A10 respectively of a 96 well plate 3 Add the 346 uL of DNA solution or DNA transfection reagent complex from step 1 to wells Al A4 A7 and A10 respectivel
12. l 1 2 Available Kits OZ Biosciences offers two types of ready to use Magnetofection reagents 1 PolyMag is a universally applicable magnetic particle preparation for high efficiency nucleic acid delivery Nucleic acids to be transfected and the magnetic particles are mixed in a one step procedure PolyMag has been used successfully with plasmid DNA phosphorothioate antisense oligonucleotides and siRNAs 2 CombiMag is a magnetic particle preparation designed to be combined with any commercially available transfection reagent such as cationic polymers and lipids and can be associated with viruses CombiMag has been used successfully with plasmid DNA antisense oligonucleotides siRNAs adenovirus and retrovirus 1 3 Kit Contents Kit contents varies according to their size e One tube containing 100 uL of particle suspension good for 100 transfections with 1 ug of DNA e One tube containing 200 UL of particle suspension good for 200 transfections with 1 ug of DNA e tube containing 1000 UL of particle suspension good for 1000 transfections with 1 ug of DNA OZ Biosciences Protocol Magnetofection www ozbiosciences com 2 Stability and Storage Storage 4 C Upon receipt and for longterm use store all reagent tubes in the fridge Magnetofection kits are stable for at least one year at the recommended storage temperature e DO NOT FREEZE THE MAGNETIC NANOPARTICLES e DO NOT ADD ANYTHING TO THE STOCK SOLUTION OF MAGNETIC NANOP
13. m Optimization Protocol O QOOOO0 210 0109 SES DYAYRADAAD HGFE DCB A 100000000 OOOOH IIGS 0000000 SOOO OOOO MIOOOOO OOO 100000000 10 11 12 A 36 iL Pal 108p 144 L PolyMag or CombiMag 3 346 pL 346 uL 346 uL 346 pL DNA or transfection complex 4 10 4 pL 6 8 pL 3 2 pL OL Serum free medium 6 180 pL 180 pL 180 pL 180 pL Serum free medium 7 Serial dilution of 180 uL Suspension cells 1 2 The composition and dilution series are performed exactly as described above from steps 1 to 5 While PolyMag CombiMag and DNA incubate step 5 above perform the following steps A Dilute the cells to be transfected to 5 x 10 1 x 10 mL in medium with or without serum and or supplement depending on cell type and cell sensitivity towards serum free conditions OZ Biosciences Protocol Magnetofection www ozbiosciences com 12 G D E Seed the cells on polylysine coated plates OR centrifuges the cells 2 minutes in order to pellet them and use the protocol for adherent cells OR Mix cell suspension with 30 uL of CombiMag reagent per 1 ml of cell suspension and follow steps C E Incubate for 10 15 minutes Distribute 100 uL of cells well of a flat bottom 96 well plate placed upon the magnetic plate Incubate for 15 minutes In the meantime continue the vector dilution series by carrying out steps 6 and 7 as above 3 Perform step 8 a
14. n is highly cell surface receptor dependent For instance adenoviruses are dependent on cells to express CAR Coxsackie s and adenovirus receptor and HIV on cells to express CD4 Unfortunately many important and interesting target tissues for fundamental research and gene therapy are non permissive to viral gene delivery tumor tissues and apical surface of lung epithelium may express variable little or none of the required receptors The association of viral vectors with CombiMag is sufficient to force infection of non permissive cells as shown with adenovirus Magnetofection also increases retroviral infectious capacity A P A s gt NIH 3T3 cells lacking CAR were infected with a recombinant adenovirus coding for LacZ CombiMag in the presence right and in the absence left of permanent magnets positioned under the culture plates Similar results were obtained with K562 cells and human peripheral blood lymphocytes Viral Magnetofection is carried out in the same manner as standard transductions with the exceptions e That virus preparations are mixed with CombiMag prior transduction e That the cell culture plate is positioned upon the magnetic plate during transduction e That polybrene or other additives are NOT used for retroviral transductions 1 Cells should be plated in the same manner as required for standard viral gene delivery For example the confluency can be high for adenoviral vectors but must be l
15. ofection www ozbiosciences com 1 1 Technology 1 1 Description Congratulations on your purchase of the Magnetofection reagent Magnetofection is a novel simple and highly efficient method to transfect cells in culture and in vivo It exploits magnetic force exerted upon gene vectors associated with magnetic particles to drive the vectors towards possibly even into the target cells In this manner the complete applied vector dose gets concentrated on the cells within a few minutes so that 100 of the cells get in contact with a significant vector dose This has several important consequences Greatly improved transfection rates in terms of percentage of cells transfected compared to standard transfections Up to several thousand folds increased levels of transgene expression compared to standard transfections High transfection rates and transgene expression levels are achievable with extremely low vector doses which allow saving expensive transfection reagents Extremely short process time in comparison to standard procedures A few minutes of incubation of cells with gene vectors are sufficient to generate high transfection efficiency Based upon a validated and recognized magnetic drug targeting technology this innovative method is e Efficient Simple amp rapid Multipurpose for all types of nucleic acid and viral amp nonwviral vectors Universal primary cells and cell lines Non toxic amp economica
16. ow for retroviral vectors which require cell division for infection Cells must be plated the day prior transfection 2 Provide a suitable amount see examples below of CombiMag in a tube large enough to contain the volume of virus preparation added in step 3 3 Add your virus preparation e g retroviral supernatant or purified adenovirus diluted in HBS PBS or cell culture medium to the tube s containing CombiMag and mix immediately by pipetting or gentle vortexing Thereafter incubate 20 minutes at room temperature 4 The ratios virus CombiMag should be adjusted according to the viral titers and cell types used For optimization we suggest as a starting point to use 1 5 UL 3 uL 6 uL and 12 uL of CombiMag with a fixed quantity of virus preparation supernatant For example For K562 adenovirus 200 MOI 6 uL of CombiMag For human PBL adenovirus 500 MOI 3 6 uL of CombiMag For NIH 3T3 adenovirus 200 MOI 3 6 UL of CombiMag For NIH 3T3 retrovirus 1 5 x 10 Xgal CFU ml 3 6 uL of CombiMag 5 Add the mixture prepared in step 3 to the cells in duplicate or triplicate 6 Place the cell culture plate upon the magnetic plate for 30 minutes OZ Biosciences Protocol Magnetofection www ozbiosciences com 9 7 Remove the magnetic plate Optionally perform a medium change 8 Cultivate the cells under standard conditions until evaluation of transgene expression 9 Depending on the viral vector
17. rotocols that were applied successfully with a variety of cell lines Optimal conditions do vary from cell line to cell line and are dependent on the nucleic acid transfection reagent or virus used Consequently the amounts and ratio of the individual components DNA and reagents may have to be adjusted to achieve best results Therefore we advise you to optimize the various transfection or infection parameters components concentration cell number incubation time Several protocol optimizations are available in the Appendix The following recommendations can be used as guidelines to achieve good transfection with minimal incubation times 4 2 General Protocol It is recommended to seed or plate the cells the day prior transfection The suitable cell density will depend on the growth rate and the conditions of the cells Cells should be 60 90 confluent at the time of Magnetofection see the suggested cell number in the table below For suspension cells use the specific protocol given below or seed the cells on polylysine coated plates 0 1 2x10 cells 96 6 well plates and use the protocol for adherent cells Immediately preceding transfection the medium can be replaced with fresh medium optionally without serum if necessary Cell Number and Transfection Volume Suggested Tissue Culture Dish Cell Number DNA Quantity Transfection ug Volume 96 well 0 5 2x10 0 1 0 5 200 uL 24 well 0 5 1x10 0 5 2 500 uL 12 well
18. rtex the tube of CombiMag Add 1 or 2 pL of CombiMag per pg of DNA to be transfected to a microtube For DNA doses of less than 1 pg predilute an aliquot of CombiMag reagent with deionized water and use the volume required for your DNA dose 2 Prepare the DNA transfection reagent complexes according to the reagent s manufacturer instructions but omit the usual final incubation step after mixing DNA amp reagent and immediately proceed to step 3 3 Add the DNA transfection reagent complex solution into the CombiMag suspension and mix immediately by vigorous pipetting 4 Incubate for 15 30 minutes 5 Add the resulting mixture to the cells to be transfected Note to transfect cells in duplicate prepare your DNA transfection reagent complexes as described above If the complexes have been prepared in 200 uL then transfer 100 uL of the resulting mixture in each well containing the cells to be transfected The total transfection volumes per well culture medium CombiMag mixture are suggested in the table above 6 Place the cell culture plate upon the magnetic plate for 5 to 20 minutes 7 Remove the magnetic plate Optionally perform a medium change 8 Cultivate the cells under standard conditions until evaluation of transgene expression 9 Depending on the commercial transfection reagent used this protocol may have to be adapted OZ Biosciences Protocol Magnetofection www ozbiosciences com 8 Virus Viral infectio
19. s above while keeping the cell culture plate on the magnetic plate 4 Continue to incubate for 15 minutes 5 Carefully remove the medium supernatant from the cells and replace with fresh complete medium while the culture plate remains positioned on the magnetic plate Be careful not to aspirate the magnetically sedimented cells 6 Remove culture plate from magnetic plate and continue to cultivate cells as desired 5 3 Quality Controls To assure the performance of each lot of Magnetofection produced we qualify each component using rigorous standards The following assays are conducted in vitro to qualify the function quality and activity of each kit component Components Standard Quality Controls PolyMag or 1 Quality and size homogeneity of the magnetic nanoparticles CombiMag 2 Stability of the magnetic nanoparticle formulations 3 Transfection efficacies on NIH 3T3 COS 7 and K562 cells Every lot shall have an acceptance specification of gt 80 of the activity of the reference lot Magnetic Plate 1 Tests of solidity 2 Test of the magnetic field force 5 4 Troubleshooting Our dedicated and specialized drug delivery systems technical support group will be pleased to answer any of your requests and to help you with your transfection experiments tech ozbiosciences com OZ Biosciences Protocol Magnetofection www ozbiosciences com 13 5 5 Bibliographic References
20. type the quantity of virus and the cell types used this protocol would have to be adjusted 4 5 Magnetofection of suspension cells 1 The composition and preparation of PolyMag DNA or CombiMag transfection reagent or virus are performed exactly as described above from steps 1 to 3 2 While PolyMag DNA or CombiMag transfection reagent or virus incubate step 4 above dilute the cells to be transfected to 5 x 10 1 x 10 mL in medium with or without serum or supplement depending on cell type and sensitivity of cells towards serum free conditions and perform one of the following three options to sediment the cells at the bottom of the culture dish in order to promote the contact with the magnetic nanoparticles a Seed the cells on polylysine coated plates and use the protocol for adherent cells OR b Briefly centrifuge the cells 2 minutes in order to pellet them and use the protocol for adherent cells OR c Mix cell suspension with 30 pL of CombiMag reagent per 1 ml of cell suspension i Incubate for 10 15 minutes ii Distribute cells to your tissue culture dish placed upon the magnetic plate volume of culture medium containing cells depends on the culture dish size see suggested transfection volume in table above as indication iii Incubate for 15 minutes 3 Add the resulting mixture of PolyMag DNA or CombiMag transfection reagent or virus to the cells while keeping the cell culture plate on the m
21. y containing PolyMag or CombiMag and mix well by pipetting 4 Fill up to 360 pL with serum and supplement free medium e g DMEM by adding 10 4 uL to Al OZ Biosciences Protocol Magnetofection www ozbiosciences com 11 10 6 8 uL to A4 and 3 2 pL to A7 Incubate for 20 30 min at room temperature In the meantime add 180 pL of serum and supplement free medium e g DMEM to the residual wells of columns 1 4 7 and 10 of the 96 well plate B1 H1 B4 H4 B7 H7 B10 H10 After the incubation in step 5 transfer 180 uL from well Al A4 A7 A10 to B1 B4 B7 B10 using a multichannel pipet mix by pipetting transfer 180 pL from B1 B4 B7 B10 to C1 C4 C7 C10 mix by pipetting from C1 C4 C7 C10 to D1 D4 D7 D10 and so on down to H1 H4 H7 H 10 Transfer 50 pL each in triplicates from column 1 to the columns 1 2 and 3 of the cell culture plate where the cells to be transfected have been seeded similarly from column 4 of the dilution plate to columns 4 5 and 6 of the culture plate from column 7 dilution plate to columns 7 8 and 9 of the culture plate and from column 10 dilution plate to columns 10 11 and 12 of the culture plate Using a multichannel pipet for the transfer Place the culture plate on the magnetic plate for 15 min Remove the magnetic plate and continue to culture cells as desired Optionally perform a medium change particularly if the transfection has been carried out in serum free mediu
22. ypes of nucleic acids delivered e Improves viral infectious capacity e Extend the host tropisms of viral vectors to non permissive cells OZ Biosciences Protocol Magnetofection www ozbiosciences com 7 Transfection Reagents A number of suppliers sell transfection reagents All of these can be associated with CombiMag reagent by simple mixing in order to generate magnetic delivery system The resulting combination leads to strong efficiency improvements for commercial transfection reagents This solution allows you to create your magnetic gene vector There are two strategies of using CombiMag One is to prepare a standard complex of DNA and a commercial transfection reagent according to the instructions of the manufacturer followed by mixing with CombiMag The second strategy is to first mix DNA and CombiMag followed by immediate mixing with the transfection reagent In this case the manufacturer s instructions are used except that instead of DNA alone a mixture of DNA and CombiMag is added to the transfection reagent Depending on the transfection reagent used the mixing order of components may influence the final transfection efficiency of Magnetofection It is recommended to use 1 or 2 pL of CombiMag per Ug of DNA in initial experiments However depending on the cell line to be transfected and the commercial transfection reagent used the optimal composition may be found above or below this ratio 1 Before each use vo
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