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1. 12w 24well NIH 3T3 200 MOI 6 12 uL 500 MOI 96 well 3x10 2x10 IU 4 Incubate 5 to 15 minutes either at room temperature or on ice 5 Add the ViroMag or ViroMag R L virus mixture to the cells to be transduced 6 Place the cell culture plate upon the magnetic plate for 15 minutes Longer incubation time 30 or 60 minutes or shorter 1 to 5 minutes for synchronization can also be used 7 Remove the magnetic plate and cultivate the cells under standard conditions until evaluation of the transduction experiment Optionally perform a medium change OZ Biosciences Protocol ViroMag www ozbiosciences com 6 4 4 Suspension Cells Protocol 1 The composition and preparation of ViroMag virus or ViroMag R L virus mixtures are performed exactly as described above from steps 1 to 4 section 4 3 pages 5 and 6 2 While the ViroMag or ViroMag R L virus mixtures incubate step 4 above prepare the cells to be transduced as suggested in Table 1 For example dilute the cells 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 cells2. 2 minutes to pellet them and use the protocol for adherent cells OR c Mix cell suspension with 20 30 uL of CombiMag reagent Magnetofection per 1 ml of cell suspension and incubate for 10 15 minutes Then distribute the cells to your tissue culture dish placed upon the magnetic plate and incubate for 15 more minutes 3 Add the resulting mixture of ViroMag or ViroMag R L virus to the cells while keeping the cell culture plate on the magnetic plate 4 Continue to incubate for 15 minutes 5 Remove culture plate from magnetic plate 6 Continue to cultivate cells as desired until evaluation of the transduction experiment 5 Appendix O 5 1 Critical Parameter for best performance 1 Cell culture conditions Best results are achieved when cells are 60 80 confluent at the time of the transduction If necessary you can wash the culture medium containing the transduction mixture after 8 24 hours and replace it by fresh medium However cells should be plated as required for standard viral gene delivery The density can be high for adenovirus but must be low for retrovirus 2 ViroMag or ViroMag R L quantity We often observed good effects at very low doses of ViroMag 2 3uL well for a 24 well plate However the efficiency may depend on the cell line and the virus type used Consequently we suggest you to start by testing a range of ViroMag volumes in order to obtain the best experimental conditions 3 Time course The infe
3. 100 of the cells get in contact with a significant vector dose ViroMag and ViroMag R L are exclusive and specific reagents dedicated to viral applications These reagents demonstrate an exceptionally high efficiency to promote control and assist viral transductions ViroMag is applicable to all viral vectors ViroMag R L is dedicated to Retrovirus Lentivirus and they present unique properties allowing to 1 Increase transduction efficiency in terms of percentage of transduced cells 2 Concentrate all viral dose on the cells very rapidly Accelerate the transduction process Infect non permissive cells Significantly improve virus infectivity with extremely low vector doses Synchronize cell adsorption infection Target confine transduction to specific area magnetic targeting ma Se Based upon a validated and recognized magnetic drug targeting technology this innovative method is e Highly Efficient Suitable for all viruses Economical Simple amp Rapid Universal primary cells hard to transfect cells and cell lines Serum compatible amp Non toxic Amenable to high throughput automation 1 2 Kit Contents Kit contents vary according to their size e 1 tube containing 0 1 mL of ViroMag or ViroMag R L good for 10 to 100 assays in a 24 well plate e 1 tube containing 0 2 mL of ViroMag or ViroMag R L good for 20 to 200 assays in a 24 well plate e 1 tube containing 1 mL of ViroMag or ViroMag R L good for 100 to 1000 ass
4. Such use is limited to the transfection and transduction of nucleic acids and virus 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 ViroMag ViroMag R L and other Magnetofection Reagents 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 ViroMag ViroMag R L and other Magnetofection Reagents material and documentation to OZ Biosciences or by destroying all ViroMag ViroMag R L and other Magnetofection Reagents components Purchasers are advised to contact OZ Biosciences with the notification that a ViroMag ViroMag R L and other Magnetofection Reagents 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 ViroMag ViroMag R L and other Magnetofection Reagents research only license and does not grant any other express or implied license The laws of the French Gove
5. with any transfection reagent for enhancing transfection efficiency 3 SilenceMag created specifically for all siRNA applications Description Reference Magnetofection Starting Kit KC30296 Magnetofection Super Starting Kit KC30496 PolyMag 200 uL PN30200 PolyMag 1000 uL PN31000 CombiMag 200 uL CM20200 CombiMag 1000 uL CM21000 SilenceMag 200 uL SM10200 SilenceMag 1000 uL SM11000 DreamFect 1 mL DF41000 DreamFect 5 x 1 mL DF45000 EcoTransfect 1 mL ET11000 VeroFect 1 mL VF61000 FlyFectin 1 mL FF51000 GeneBlaster Ruby GB20011 GeneBlaster Sapphire GB20012 GeneBlaster Topaz GB20013 B Galactosidase ONPG assay kits GO10001 B Galactosidase CPRG assay kits GC10002 X Gal staining kit GX10003 Contain PN30100 CM20100 MF10096 Contain PN30100 CM20100 SM10200 MF10096 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 ViroMag www ozbiosciences com 8 Purchaser Notification Limited License The purchase of the ViroMag ViroMag R L and other Magnetofection Reagents grants the purchaser 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
6. 10 0 5 1x10 150 uL 24 well 0 5 1x10 2 5x10 500 uL 12 well 1 2x10 2 5 10x10 1 mL 6 well 2 5x10 1 2x10 2 mL 60 mm dish 5 10x10 2 5 5x 10 4 mL 90 100 mm dish 15 30x10 5 10x10 8 mL T 25 flask 5 10x10 2 5 5x10 5 mL T 75 flask 20 50x 10 5 15x10 10 mL Transduction volume corresponds to the volume of culture medium covering the cells plus the volume of the ViroMag virus mixture According to the standard protocol the virus ViroMag or ViroMag R L mixtures are prepared in medium with or without serum and supplement or in physiological saline These mixtures are then added to the cells that are covered with complete medium Therefore the addition of this cocktail will result in the dilution of supplements such as serum antibiotics or other additives of your standard culture medium if medium without serum and supplement or physiological saline is used Although a medium change after Magnetofection is not required for most cell types it may be necessary for cells that are sensitive to serum supplement concentration If cell culture viral supernatant is used instead you can also replace the cell culture medium by that one 4 3 ViroMag Procedure Viral Magnetofection is carried out in the same manner as standard transductions with the following exceptions e Virus preparations are mixed with ViroMag or ViroMag R L prior to transduction e Cell culture plate is positioned upon the magnetic plate during transduction e Polybren
7. Magnetofection ViroMag amp ViroMag R L oO SS OZ BIOSCIENCES ViroMag amp ViroMag R L Instruction Manual ViroMag is a superior reagent based on the Magnetofection technology suitable for all viral applications ViroMag R L is an improved ViroMag formulation specifically designed for Retrovirus Lentivirus vectors List of ViroMag and ViroMag R L Kits Catalog Description Volume pL Number of Number of Number transductions transductions 24 well plates 96 well plates VM40100 ViroMag 100 100 10 100 30 500 VM40200 ViroMag 200 200 20 200 60 1000 VM41000 ViroMag 1000 1000 100 1000 300 5000 RL40100 ViroMag R L 100 100 10 100 30 500 RL40200 ViroMag R L 200 200 20 200 60 1000 RL41000 ViroMag R L 1000 1000 100 1000 300 5000 MF10096 Magnetic Plate with 96 magnets MF10000 Super Magnetic Plate gt KC30500 ViroMag Starting Kit 200 20 200 60 1000 KC30596 ViroMag Starting Kit 7 200 20 200 60 1000 t Contains 1 vial of ViroMag VM40200 and a Super Magnetic Plate MF10000 Contains 1 vial of ViroMag VM40200 and a Magnetic Plate with 96 magnets 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 For all other supplementary information do not hesitate to contact our dedicated technical support tech ozbiosciences com OZ BIOSCIENCES Parc Scientifique et Technologique de Luminy BP13 13273 Marseille Cedex 9 Fran
8. a fixed quantity of virus preparation supernatant in 24 well Refer to Table 2 for the other ranges of dose 3 Add your virus preparation to the tube s containing ViroMag or ViroMag R L and mix immediately by pipetting up and down Virus preparation is preferably in serum free medium or salt containing buffers Note 1 If required dilute the aliquot of your virus preparation to be used for transduction with serum free cell culture medium or other salt containing buffer e g retroviral supernatant or purified adenovirus diluted in HBS PBS or cell culture medium Alternatively you can directly use an aliquot of culture supernatant from a producer cell line Note 2 The ratios virus ViroMag should be adjusted according to the viral titers and cell types used Table2 Recommended amount of ViroMag amp ViroMag R L volume of vector preparation and final transduction volume Culture Vessel ViroMag Suggested Volume of Final Transduction Quantity ViroMag ViroMag virus Volume oe Quantity pL solution 96wel ft 02 3 50 uL 150 uL 24 well oo 1 2 0 100 uL 500 uL 12 well 100 uL 60mmdish 10 120 Om T 25 flask 10 120 OL Pm Transduction volume corresponds to the volume of culture medium covering the cells plus the volume of the ViroMag virus mixture Table3 Successful examples of ViroMag and ViroMag R L experimental procedure Cell types Virus type Titer ViroMag Culture MOL CFU TCIDso Quantity EE EEE K562 200 MOI
9. agnetic influence and a specific targeting to define area can be done Indeed magnetic targeting confine to specific area linked to the magnet size and shape has been demonstrated for adenovirus AAV baculovirus and retrovirus Bibliographic References 1 Scherer F et al Magnetofection enhancing and targeting gene delivery by magnetic force in vitro and in vivo Gene Ther 2002 9 2 102 9 Plank C et al Enhancing and targeting nucleic acid delivery by magnetic force Expert Opin Biol Ther 2003 3 5 745 58 Schillinger U et al Advances in Magnetofection magnetically guided nucleic acid delivery 2005 J Magn Magn Mat 293 501 508 Mok H et al Evaluation of polyethylene glycol modification of first generation and helper dependent adenoviral vectors to reduce innate immune responses 2005 Mo Ther 11 1 66 79 5 Pandori M W et al Adenovirus Microbead Conjugates Prossess Enhanced Infectivity A New Strategy to Localized Gene Delivery 2002 Virology 299 204 212 6 Mah C et al Improved Method of Recombinant AAV2 Delivery for Systemic Targeted Gene Therapy 2002 Mol Ther 6 1 106 112 7 Haim H et al I and Panet A Synchronized infection of cell cultures by magnetically controlled virus 2005 J Viro 79 1 622 5 8 Tai MF et al Generation of magnetic retroviral vectors with magnetic nanoparticles 2003 Rev Adv Mater Sci 5 319 323 9 Hughes C et al Streptavidin paramagnetic particles provi
10. ata quickly and if necessary we advise you to optimize the experimental conditions parameters in order to achieve the best effects Optimal conditions do vary from cell to cell and are highly dependent upon the type of virus used its titer the composition of the viral solution and cell culture conditions Consequently the amount concentration and ratio of the individual components virus and ViroMag the time course and the number of cells may have to be adjusted to get the best results Several optimization protocols are available in the Appendix 4 2 Cell Culture It is recommended to seed or plate the cells the day prior transduction however cells can also be prepared few hours before the transduction Suspension cells should be prepared in the adequate vessel just before the infection see below for specific protocol The suitable cell density will depend on the growth rate and the condition of the cells Best results are achieved if cells are at least 60 80 confluent at the time of Magnetofection see the suggested cell number in the table below 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 low for retroviral vectors which require cell division for infection Table 1 Recommended cell number Culture vessel Number of adherent cells Number of suspension cells Final Transduction Volume 96 well 0 05 0 15 x
11. ays in a 24 well plate Stability and Storage Storage 4 C Upon receipt and for long term use store all 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 DO NOT ADD ANYTHING TO THE STOCK SOLUTION OF MAGNETIC NANOPARTICLES Shipping condition Room Temperature OZ Biosciences Protocol ViroMag www ozbiosciences com 2 2 Applications O 2 1 Cell Types ViroMag and ViroMag R L are generally applicable on numerous cell types This technology has been tested successfully on a variety of immortalized and primary cells If a particular cell type is not listed this does not imply that these reagents are not going to work OZ Biosciences is maintaining an updated list of cells successfully tested available on the website www ozbiosciences com Cell Line Cell Type Source 293 HEK 293 Transformed embryonic kidney ee 293 T EBNA 181RDB Pancreatic cells o Human B lymphoblastoid Simian Marmoset eee COLO 205 Colon adenocarcinoma Fibroblast like kidney para Osteosarcoma amine Meta Cervical epithelial carcinoma Human Endothelial cells primary Myelogenous leukemia sts Fbroblasts CT Peiz pheochromocytoma adrenal Rat ova ovarian carcinoma Human Fibroblast kidney Green Monkey PrimaryKeratinocytes Human Mouse Primary peripheral blood ymphocytes Human Mouse Primarymusciecets House 2 2 Types
12. ce Tel 33 0 491 828 175 Fax 33 0 491 828 170 E mail contact ozbiosciences com Web Site www ozbiosciences com Patent Pending OZ BIOSCIENCES The art of delivery systems OZ Biosciences Protocol ViroMag www ozbiosciences com 1 1 Technology O 1 1 Description Congratulations on your purchase of the ViroMag and or ViroMag R L reagent ViroMag is a specific formulation issued from our Magnetofection technology designed to be used in association with all viral vectors and for many transduction applications ViroMag R L is an optimized nanoparticles formulation specifically developed for Retrovirus Lentivirus For the first time scientists will be able to increase transduction efficiency infect non permissive cells concentrate virus onto cells or in culture medium accelerate infection process or synchronize infection without modification of the viruses just by associating ViroMag or ViroMag R L reagents to the viral vectors ViroMag and ViroMag R L are the only reagents available offering a solution to such applications Magnetofection is a novel simple and highly efficient viral and non viral gene delivery method It exploits magnetic force exerted upon gene vectors associated with magnetic particles to drive the nucleic acids or virus towards possibly even into the target cells In this manner the complete applied nucleic acid and viral dose gets concentrated on the cells within a few minutes so that
13. ction time course depends on the amount concentration of virus used Indeed longer incubation under the magnetic field is required with very low viral titers whereas with high viral dose short incubation times are sufficient 5 2 Protocol Optimization In order to get the best out of ViroMag and ViroMag R L several parameters can be optimized e ViroMag dose amp Ratio of ViroMag to Virus e Cell type cell density and incubation times 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 ViroMag or ViroMag R L dose with a fixed amount of virus This will vary the concentration of ViroMag and the ratio ViroMag Virus To this end vary the amount of ViroMag in the range suggested in the Table 2 For instance from 0 2 to 3uL of ViroMag or ViroMag R L in a 96 well plate 2 Next you can inverse the procedure by optimizing the dose of virus with a fixed amount of reagent 3 After having identified the correct quantity of ViroMag or ViroMag R L and virus you could pursue the process by optimizing the cell number density and time course of incubation between ViroMag and viruses section 4 3 4 and under the magnetic plate section 4 3 6 OZ Biosciences Protocol ViroMag www ozbiosciences com 7 5 3 Quality Contr
14. de a choice of three affinity based capture and magnetic concentration strategies for retroviral vectors 2001 Mo Ther 3 4 623 30 10 Kadota S I et al Enhancing of measles virus infection by magnetofection 2005 J Virol Methods 11 Raty J K et al Enhanced gene delivery by avidin displaying baculovirus 2004 Mo Ther 9 2 282 91 12 Satoh et al Virus concentration using polyethyleleimine conjugated magnetic beads for improving the sensitivity of nucleic acid amplification tests J Virol Methods 114 11 19 e N As for all Magnetofection reagents ViroMag and ViroMag R L require an appropriate magnetic field Two magnetic plates 96 magnets plate and Supermagnetic plate especially designed for Magnetofection to exert this specific magnetic field are available Their special geometry produce a strong magnetic field that is suitable for all cell culture dishes T 75 flasks 60 amp 100 mm dishes 6 12 24 48 and 96 well plates OZ Biosciences Protocol ViroMag www ozbiosciences com 4 4 Protocol 4 1 General Considerations The instructions given below represent sample protocols that were applied successfully with a variety of cells and viruses Our R amp D team has tested and optimized the ViroMag and ViroMag R L reagents in order to provide you with the straightforward and efficient procedure Therefore we recommend you to start by following our general protocol as guidelines to obtain good d
15. e or other additives are NOT used for transductions The protocol is straightforward For instance 30 uL of ViroMag magnetic particles have been found sufficient to bind 10 billion of viral particles Thus the particle amounts listed in Table 2 will be mainly sufficient to bind virus doses which are usually applied in transduction experiments Depending on the viral vector type the quantity of virus and the cell type used this protocol would have to be adjusted see appendix for optimization protocol ViroMag R L is an improved formulation of ViroMag specifically designed for Retrovirus and Lentivirus and should be used the same way as ViroMag OZ Biosciences Protocol ViroMag www ozbiosciences com 5 1 Plate the cells the day before infection or just before infection in your appropriate tissue culture dish as suggested in Table 1 2 Add a suitable amount see table below of ViroMag or ViroMag R L in a tube large enough to contain the volume of virus preparation added in step 3 If required ViroMag amp ViroMag R L can only be diluted with deionized water Do not dilute the reagents in serum and supplement free medium The amount of ViroMag or ViroMag R L depends on the type and dose of virus used As a starting point the suggested ViroMag quantity indicated in the table 2 can be used However we highly recommend adjusting the amount of ViroMag For example use 1 5 uL 3 uL 6 uL and 12 uL of ViroMag or ViroMag R L with
16. eases transduction efficiency The combination of paramagnetic nanoparticles with adenovirus has shown up to 500 fold enhancement of gene expression compared with standard infection Significant enhancement up to 70 fold of the infection of measles virus has been reported as well as for HIV and VSV about 100 fold increase v concentrates viral dose promotes and accelerates the infection process Retroviral titers could be increased by 1000 to 4000 fold Concentration of measles virus aav non enveloped virus SV40 and enveloped virus such as Sindbis virus HSV I and VSV has been reported Transduction efficiency of PEGylated adenovirus can be restored by the use of magnetic nanoparticles v improves viral infectious capacity Significant enhancement of retrovirus infectivity can be achieved with the use of magnetic nanoparticles v extends the host tropisms of viral vectors to non permissive cells gt The association of viral vectors with magnetic nanoparticles is sufficient to force infection of non permissive cells as shown with adenovirus in NIH 3T3 K562 cells human peripheral blood lymphocytes COLO25 and C6 and with the measles virus in SLAM negative cell lines v allows the synchronization of the transduction Synchronized adsorption of HIV 1 on primary cells can be accomplished with the use of magnetic nanoparticles v can provide a magnetic targeting 04t High transduction can be achieved under m
17. of Virus ViroMag reagent can usually be combined with any viruses ViroMag R L is particularly suitable for Lentivirus Retrovirus If a particular virus is not listed this does not imply that these reagents are not going to work OZ Biosciences is maintaining an updated list of virus successfully tested that is available on the website www ozbiosciences com Virus Type Virus name Application Adenovirus Ad5 LacZ Ad5 PEG Increase transduction infect non permissive cells Adeno Associated Virus Increase transduction infect non permissive cells Lentivirus Retrovirus HIV MuLV MLV FIV Increase infectivity synchronize infection Herpes virus HSV I Concentration Alpha virus Sindbis virus Concentration Baculovirus Baavi Increase transduction targeting Rhabdovirus VSV Concentration Polyomavirus SV40 Concentration Paramyxovirus Measles Increase transduction infect non permissive cells OZ Biosciences Protocol ViroMag www ozbiosciences com 3 2 3 Application examples amp Bibliographic References Until now a universal method enhancing assisting controlling and promoting viral gene delivery systems was lacking Magnetofection is the only existing method answering these different needs Many studies have demonstrated the potential of using Magnetic Particles such as ViroMag and ViroMag R L for viral applications The conducted studies have shown that magnetic particles including ViroMag and ViroMag R L v incr
18. ols To assure the performance of each lot of ViroMag amp ViroMag R L produced we qualify each lot using rigorous standards n vitro assays are conducted to qualify the quality and activity of each kit component Standard Quality Controls ViroMag amp 1 Quality and size homogeneity of the magnetic nanoparticles ViroMag R L Stability of the magnetic nanoparticles formulations ViroMag transduction efficacies with a recombinant adenovirus on NIH 3T3 cells Every lot shall have an acceptance specification of gt 80 of the activity of the reference lot 4 ViroMag R L transduction efficacies with a recombinant pseudo HIV GFP on HeLa cells Every lot shall have an acceptance specification of gt 80 of the activity of the reference lot Sterility Thioglycolate assay absence of fungal and bacterial contamination shall be obtained for 7 days Magnetic Plate 1 Tests of solidity and Test of the magnetic field force 5 4 Troubleshooting Our dedicated and specialized technical support team will be pleased to answer any of your requests and to help you with your transfection experiments at tech ozbiosciences com In addition do not hesitate to visit our website www ozbiosciences com and the FAQ section 6 Related Products OZ Biosciences offers three other types of ready to use Magnetofection reagents 1 PolyMag designed for all transfection applications and all nucleic acids 2 CombiMag aimed to be combined
19. rnment shall govern the interpretation and enforcement of the terms of this License Product Use Limitations The ViroMag ViroMag R L and other Magnetofection Reagents 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 ViroMag www ozbiosciences com 9
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