PolyMag/Neo & CombiMag
Contents
1. perform a serial dilution of a preformed magnetic vector complex 3 After having identified the correct quantity of CombiMag PolyMag or PolyMag Neo nucleic acid transfection reagent commercial 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 supplement free medium e g DMEM 2 Provide 3 6 7 2 10 8 and 14 4 uL respectively of Po vMag or PolyMag Neo in case of DNA or CombiMag in case of DNA transfection reagent complex in well A1 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 A1 A4 A7 and A10 respectively containing Po yMag PolyMag Neo or CombiMag and mix well by pipetting 4 Fill up to 360 uL with serum and supplement free medium e g DMEM by adding 10 4 uL to Al 6 8 uL to A4 and 3 2 uL to A7 1 11 12 0000000 OOOO OON Y DAIO OOOO S 0000000 wO OJIJI G OOO0O0O0OO Olja 000000080 OOOOOOOO 2S ES Ce DOO OOOO 9 O2. Magnetofection PolyMag Neo amp CombiMag PRIMARY CELLS CELL LINES OZ BIOSCIENCES Magnetofection Po yMag Neo amp CombiMag Instruction Manual Magnetofection is a novel simple and highly efficient 7 vitro and in vivo transfection method List of Magnetofection Kits Catalog Description Volume pL Size number of Number of Number transfections ug transfections of DNA 96 well plates PN30100 PolyMag reagent 100 100 1000 PN30200 PolyMag reagent 200 200 2000 PN31000 PolyMag reagent 1000 1000 10000 PG60100 PolyMag neo reagent 100 100 1000 PG60200 PolyMag neo reagent 200 200 2000 PG61000 PolyMag neoreagent 1000 1000 10000 CM20100 CombiMag reagent 100 100 1000 CM20200 CombiMag reagent 200 200 2000 CM21000 CombiMag reagent 1000 1000 10000 KM30200 Selection Kit 3 x 100 200 2000 KM30300 Selection Kit siP 200 2 x 100 400 1000 KM30350 Selection Kit siC 200 100 400 1000 KM30400 Super Selection Kit 200 3 x 100 200 400 2000 KC30200 Magnetofection Starting Kit gt 3 x 100 200 2000 KC30400 Super Starting Kit 200 3 x 100 200 400 2000 KC30296 Magnetofection Starting Kit 3 x 100 200 2000 KC30496 Super Starting Kit 200 3 x 100 200 400 2000 MF10000 Super Magnetic Plate N A N A N A MF14000 Mega Magnetic Plate N A N A N A MF10096 96 Magnets Magnetic Plate N A N A N A t Contains 1 vial of each reagent Po yMag PolyMag Neo and CombiMag Contains 1 vial of each reagent Si
3. O O OC O OC O 9 1 10 11 12 2 3 6uL 7 2 uL 10 8 uL 14 4uL 3 346uL 346 UL 346 uL 346 uL 4 10 4uL 6 8 uL 3 2 uL 0 uL 6 180pL 180 uL 180 uL 180 uL 2 5 Incubate for 20 30 min at room temperature 6 Inthe meantime add 180 uL 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 7 After the incubation in step 5 transfer 180 uL from well A1 A4 A7 A10 to B1 B4 B7 B10 using a multichannel pipet mix by pipetting transfer 180 uL from B1 B4 B7 B10 to C1 C4 C7 C10 mix by pipetting from C1 C4 C7 C10 to Di D4 D7 D10 and so on down to H1 H4 H7 H10 8 Transfer 50 uL 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 OZ Biosciences Protocol Magnetofection www ozbiosciences com 8
4. e 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 Optionally perform a medium change Remove the magnetic plate For viral application we recommend the use of ViroMag or ViroMag R L Note For certain cells primary cells such as neurons a medium change at this step significantly improves the transfection efficiency and greatly minimizes potential cytotoxicity To process the medium change leave the cells onto the magnetic plate remove the culture medium and replace it with fresh culture complete medium Thereafter remove the magnetic plate and continue to step 8 below 1 Cultivate the cells under standard conditions until evaluation of transgene expression 2 Depending on the commercial transfection reagent used this protocol may have to be adapted For viral application we recommend the use of ViroMag or ViroMag R L OZ Biosciences Protocol Magnetofection www ozbiosciences com 6 4 5 Magnetofection of suspension cells 1 The composition and preparation of Po vMag or PolyMag Neo DNA or CombiMag transfection reagent are performed exactly as described above from steps 1 to 3 2 While PolyMag or PolyMag Neo DNA or CombiMag transfection reagent are incubating 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 cel
5. e transfected with a commercial incubated with a commercial transfection transfection reagent L CombiMag with reagent G CombiMag with and without the and without the magnetic field for 15 min magnetic field for the indicated time spans Luciferase expression was assayed after 24h Luciferase expression was assayed after 24h OZ Biosciences Protocol Magnetofection www ozbiosciences com 3 2 2 Nucleic Acids Types and Vectors The CombiMag reagent can be combined with any nucleic acid and all transfection reagents Nucleic Acid or Virus PolyMag CombiMag SilenceMag ViroMag PolyMag Neo DNA plasmid Antisense Oligonucleotides mRNA siRNA Viruses 2 3 Cell Types Magnetofection is applicable with numerous cell types and has been successfully tested on a variety of immortalized cell lines as well as primary cells see list on website If a particular cell type or cell line is not listed this does not imply that Magnetofection is not going to work An updated list of cells successfully tested as well as product citations 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 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 f
6. enceMag PolyMag and PolyMag Neo 3 Contains 1 vial of each reagent Si lenceMag and CombiMag Contains 1 vial of each reagent Si enceMag PolyMag PolyMag Neo amp CombiMag gt Contains 1 vial of each reagent Po yMag PolyMag Neo and CombiMag plus a Super Magnetic Plate MF10000 Contains 1 vial of each reagent Si enceMag PolyMag PolyMag Neo amp CombiMag plus a Super Magnetic Plate MF10000 Contains 1 vial of each reagent Po yMag PolyMag Neo and CombiMag plus a 96 magnets Magnetic Plate MF10096 8 Contains 1 vial of each reagent Si enceMag PolyMag PolyMag Neo amp CombiMag plus a 96 magnets Magnetic Plate MF10096 Use the content of the table above to determine the appropriate catalog number for your needs You can order these products by contacting us phone fax email website For all other supplementary information do not hesitate to contact our dedicated technical support tech ozbiosciences com and or to visit our website www ozbiosciences com OZ BIOSCIENCES Parc Scientifique et Technologique de Luminy Zone entreprise case 922 13288 Marseille Cedex 9 France Tel 33 0 4 86 94 85 16 Fax 33 0 4 86 94 85 15 E mail contact ozbiosciences com Site Internet www ozbiosciences com OZ Biosciences Protocol Magnetofection www ozbiosciences com 1 Table of Contents 1 Technology 1 1 Description L2 Available Reagents 13 Kit Contents 2 Applications Zl Nucleic Acids Dose Respo
7. ields Its special geometry not only produces strong magnetic fields under each well of 96 well plates but is also applicable for other plate formats T 75 flasks 60 amp 100 mm dishes 6 12 and 24 well plates Super Magnetic Plate suits for all cell culture supports and Mega Magnetic Plate is designed to hold up to 4 culture dishes at one time The magnetic plate design allows producing a heterogeneous magnetic field that magnetizes the nanoparticles in solution forms a very strong gradient and covers all the surface of the plate Magnetic plate 96 magnets Super Magnetic Plate Mega Magnetic Plate OZ Biosciences Protocol Magnetofection www ozbiosciences com 4 4 Protocols O 4 1 General Considerations Instructions given below represent sample protocols that were successfully applied with a variety of cell lines Optimal conditions do vary from cell line to cell line and are dependent on 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 and upon request by email The following recommendations can be used as guidelines to achieve good transfection with minimal incubation time
8. l 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 to pellet them and use the protocol for adherent cells OR c Mix cell suspension with 30 uL of CombiMag reagent per mL of cell suspension i Incubate for 10 15 minutes li 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 OR d Incubate the cells in serum free medium during 2 hours prior Magnetofection The absence of serum allows some cells to adhere onto the plastic dish surface 3 Add the resulting mixture of Po vMag or PolyMag Neo DNA or CombiMag transfection reagent to the cells while keeping the cell culture plate on the magnetic plate 4 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 desi
9. n reagents 4 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 e Multipurpose for all types of nucleic acids and non viral vectors e Universal primary cells and cell lines e Non toxic amp economical OZ Biosciences Protocol Magnetofection www ozbiosciences com 2 1 2 Available Reagents OZ Biosciences offers three types of ready to use Magnetofection transfection 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 antisense oligonucleotides and siRNA 2 PolyMag Neo is an optimized formulation of PolyMag for a higher gene expression level in primary hard to transfect and cell lines 3 CombiMag is a magnetic particle preparation designed to be combined with any commercially available transfection reagent such as cationic polymers and lipids CombiMag has been used successfully with plasmid DNA antisense oligonucleotides mRNA and siRNA Other Magnetofection based reagents have been developed by OZ Biosciences SilenceMag specifically designed for
10. nse and Transfection Kinetics 2 2 Nucleic Acids Types and Vectors 2 3 Cell Types 3 Magnetofection Apparatus 4 Protocols 4 1 General Considerations 4 2 General Protocol 4 3 PolyMag or PolyMag Neo 4 4 CombiMag I Ja i U1 ODMDDNAKHAAUUNUNARAARWW 09 4 5 Magnetofection of Suspension Cells 5 Appendix 11 Sel Protocol Optimization 5 2 Protocol Optimization in a 96 well format 5 3 Quality Controls 10 5 4 Troubleshooting 11 6 Related Products 11 7 Purchaser Notification 11 1 Technology 1 1 Description Congratulations on your purchase of the Magnetofection reagent Magnetofection is an original 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 1 Greatly improved transfection rates in terms of percentage of cells transfected compared to standard transfections 2 Up to several thousand folds increased levels of transgene expression compared to standard transfections 3 High transfection rates and transgene expression levels are achievable with extremely low vector doses which allow saving expensive transfectio
11. pt 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 uL 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 vortex the tube of CombiMag Add 1 or 2 uL of CombiMag per ug of DNA to be transfected to a microtube For DNA doses of less than 1 ug predilute an aliquot of Comb Mag 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 Comb Mag 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 volum
12. red until evaluation of transgene expression 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 Magnetofection Several parameters can be optimized e Nucleic acid dose used e Ratio of CombiMag PolyMag or PolyMag Neo to nucleic acid e 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 to optimize one parameter at a time and start from the experimental procedures described above in section 4 1 Start by optimizing the ratio Po yMag or PolyMag Neo DNA or CombiMag transfection reagent To this end use a fixed amount of DNA Vary the amount of CombiMag or PolyMag or PolyMag Neo from 0 25 to 5uL ug of DNA The ratio Po yMag PolyMag Neo or CombiMag DNA can be changed by doubling or multiplying the volumes of the reagents used Reagents can be pre diluted 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 OZ Biosciences Protocol Magnetofection www ozbiosciences com 7 2 Thereafter change the nucleic acid dose with a fixed ratio of Po yMag or PolyMag Neo DNA or CombiMag transfection reagent that has been previously optimized For this purpose you can
13. s 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 cells conditions 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 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 2x 10 0 1 0 5 200 uL 24 well 0 5 1x10 0 5 2 500 uL 12 well 1 2x10 2 4 1 mL 6 well 2 4x10 2 6 2 mL 60 mm dish 5 10x10 6 8 4 mL 90 100 mm dish 10 20x 10 8 12 8 mL T 75 flask 20 50 x 10 10 20 12 mL The same protocol can be used to produce stably transfected cells except that 48 hours post transfection fresh medium containing the appropriate antibiotics are transferred to cells for selection It is important to wait at least 48 hours before exposing the transfected cells to selection media Vectors are prepared in medium without serum and supplement or in physiological saline buffer According to the standard Magnetofection protocol the serum and supplement free vector cocktail is added to the cells that are cultured in complete medium Therefore the addition of the transfection solution will result in the dilution of s
14. siRNA delivery ViroMag amp ViroMag R L specially developed for all viral applications and NeuroMag created to achieve efficient transfection of primary neurons and neuronal cell lines Further detailed information on these specifics reagents can be found at www ozbiosciences com 1 3 Kit Contents Kit contents differ according to their size 1 tube containing 100 uL of particle suspension good for 100 transfections with 1 ug of DNA 1 tube containing 200 uL of particle suspension good for 200 transfections with 1 ug of DNA 1 tube containing 1000 uL of particle suspension good for 1000 transfections with 1 ug of DNA Stability and Storage Storage 4 C Upon receipt and for long term 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 DONOT ADD ANYTHING TO THE STOCK SOLUTION OF NANOPARTICLES Shipping condition Room temperature 2 Applications O 2 1 Nucleic Acids Dose Response and Transfection Kinetics Save Materials Save Times 200 paramagnetic vehicle plus magnetic field gt g g z 150 paramagnetic vehicle z no magnetic field WY WY 100 D 2 standard gene transfer A 2 S04 z Cc O O Q Q no N Hi o e 0 0 05 0 1 0 15 0 50 100 150 200 250 dose ug DNA process time min DNA dose response profile NIH 3T3 cells Transfection kinetics NIH 3T3 cells were wer
15. tandard culture medium For most cell types a medium change is not required after Magnetofection However 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 up to 4 hours In this case a medium change will be required after Magnetofection 4 3 PolyMag or PolyMag Neo The protocol is as simple as follows use 1 uL of PolyMag or PolyMag Neo per ug of DNA 1 Before each use vortex the Po yMag or PolyMag Neo material Add 1 to 10 uL of PolyMag or PolyMag Neo 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 predilute Po yMag or PolyMag Neo 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 Pol yMag or PolyMag Neo 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 Po yMag or PolyMag Neo mixture are suggested in the table above Note to transfect cells in duplicate prepare your DNA Po yMag or PolyMag Neo complexes as described previously and transfer 100 uL of the resulting mixture to each well containing the cells to be transfected OZ Biosciences Protocol Magnetofection w
16. ww ozbiosciences com 5 5 Place the cell culture plate upon the magnetic plate for 5 to 20 minutes 6 Optionally perform a medium change Remove the magnetic plate 7 Cultivate the cells under standard conditions until evaluation of transgene expression For siRNA application we recommend the use of SilenceMag 4 4 CombiMag Until now a universal method enhancing the efficiency of synthetic non 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 types of nucleic acids delivered A number of suppliers sell transfection reagents They all can be associated with CombiMag by simple mixing in order to generate magnetic delivery system The resulting mixture 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 exce
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