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3D-Fect

1. 3D Transfection Reagent for Scaffolds Sponges Matrices Inserts 3D Fect Instruction Manual 3D Fect transfection reagent 3D Transfection a new outlook for your cells 3D Fect is the latest OZ Biosciences reagent for 3D transfection It has been specifically designed and developed for transfection of cells cultured in 3D scaffolds sponges matrices inserts This reagent is based on a novel technology that allows adding a third dimension to cell cultures List of 3D Fect Kits Catalog Description Volume pL Size number of Number transfection ug of DNA TF20250 3D Fect 250 65 TF20500 3D Fect 500 125 TF21000 3D Fect 1000 250 Use the content of the table above to determine the appropriate catalog number for your needs You can order these products by contacting us telephone fax mail e mail or directly through our website For all other supplementary information do not hesitate to contact our dedicated technical support tech ozbiosciences com OZ Biosciences Parc Scientifique de Luminy Zone Luminy Entreprise Case 922 13288 Marseille Cedex 9 FRANCE http www ozbiosciences com Tel 33 0 4 86 94 85 16 Fax 33 0 4 86 94 85 15 E mail contact ozbiosciences com Web Site www ozbiosciences com OZ BIOSCIENCES The art of delivery systems OZ Biosciences Protocol 3DFect vs 1 6 www ozbiosciences com 1 Table of Contents 1 Technology 2 1 1
2. The optimal time range between transfection and assay for gene activity varies with cell line promoter activity expression product etc The transfection efficiency can be monitored after 1 to several days Reporter genes such as GFP B galactosidase secreted alkaline phosphatase or luciferase can be used to quantitatively measured gene expression These control plasmids completely compatible and successfully tested with 3D Fect reagent are available at www ozbiosciences com pVectOZ GFP LacZ Luc SEAP CAT OZ Biosciences team has developed a detailed protocol for optimization and also cell specific optimal transfection procedures Thus do not hesitate to contact our technical service at tech ozbiosciences com to request these specific protocols OZ Biosciences Protocol 3DFect vs 1 6 www ozbiosciences com 6 4 Appendix 4 1 Quality Controls To assure the performance of each lot of 3D Fect produced we qualify each component using rigorous standards The following jn vitro assays are conducted to qualify the function quality and activity of each kit component Specification Standard Quality Controls Purity Silica Gel TLC assays Every compound shall have a single spot Sterility Thioglycolate assay Absence of fungal and bacterial contamination shall be obtained for 7 days Biological Activity Transfection efficacies on NIH 3T3 and COS 7 cells Every lot shall have an acceptance specification of
3. Description 2 1 2 Kit Contents 2 2 Applications 3 2 1 Application Areas 3 2 2 Cell Types 3 3 General Protocols 3 6 3 1 General Considerations 3 3 2 Cell Preparation 3 3 3 Protocol for 3D Scaffold 4 3 4 Optimization Protocol 5 6 4 Appendix 7 8 4 1 Quality Controls 7 4 2 Troubleshooting 7 8 5 Related Products 9 6 Purchaser Notification 10 1 Technology O 1 1 Description Congratulations on your purchase of our latest 3D Fect transfection reagent 3D Fect is our newest transfection reagent specifically designed and developed for cell cultured on 3D Scaffolds 3D matrices not only add a third dimension to cells environment they also allow creating significant differences in cellular characteristics and behavior Because 3D scaffolds are routinely used in basic research and therapeutic applications OZ Biosciences has developed two new powerful reagents 3D FectIN for hydrogels and 3D Fect for scaffolds In this way 3D matrices bearing complexes formed with 3D Fect reagent are colonized by cells to be transfected in a more natural environment 3D Fect reagent associated with 3D matrices allows numerous cell transfections in order to follow tissue engineering tissue regeneration tumor invasion neural differentiation cellular polarization tissue formation colonization neurite growth Principal 3D Fect advantages Highly efficient Ideal for any 3D scaffolds sponges matrices
4. Protocol 3DFect vs 1 6 www ozbiosciences com 5 1 3D Fect DNA ratio This is an important optimization parameter Depending on the 3D matrix 3D Fect reagent has to be used in slight excess compare to DNA but the optimal ratio will also depend on the cells used For optimization first maintain a fixed quantity of DNA according to the size of your scaffold or cell number and then vary the amount of 3D Fect reagent over the suggested range in the Table 2 You can test ratios from 1 to 6 uL of 3D Fect reagent per 1 ug DNA Table 2 Suggested range of 3D Fect for 3D Fect DNA ratio optimization Scaffold Size DNA 3D Fect 3D Fect Volume Hg Volume pL HL proposed interval 0 05 cm 0 5 x 0 5 x 0 2 1 1 6 1 2 3 4 5 6 0 125 cm 0 5 x 0 5 x 0 5 3 3 18 3 6 9 12 15 18 0 5 cm 1x 1x 0 5 15 15 90 15 30 45 60 75 90 2 Quantity of DNA After optimization of the 3D Fect DNA ratio proceed to adjust the best amount of DNA by maintaining a fixed ratio of 3D Fect to DNA and vary the DNA quantity over the suggested range Table 3 Table 3 Suggested range of DNA amounts for optimization with 3D Fect Scaffold Size DNA DNA quantity ug _ proposed interval 0 05 cm 0 5 x 0 5 x 0 2 0 5 2 0 5 1 1 5 2 0 125 cm 0 5 x 0 5 x 0 5 1 5 6 1 5 3 4 5 6 0 5 cm 1x 1x 0 5 7 5 30 7 5 15 22 5 30 Thereafter cell number culture medium compositions incubation times can als
5. and includes a positive control 13 3D Fect reagent temperature Reagents should have an ambient temperature and be vortexed prior to use 14 Transfection reagent storage Transfection efficiency can slowly decrease if 3D Fect is kept more than one week at RT Store at 4 C to recover initial efficiency Cellular toxicity 1 Unhealthy cells 1 Check cells for contamination 2 Use new batch of cells 3 Ensure culture medium conditions pH type of medium used contamination etc 4 Cells are too confluent or cell density is too low 5 Verify equipments and materials 6 ensure compatibility of 3D matrices with cell type 2 Matrix Composition Ensure that Matrices are compatible with the cells depending on their compositions 3D scaffolds will allow cell to attach or not non adhered cells will go on apoptosis 3 Transgene product is toxic Use suitable controls such as cells alone transfection reagent alone or mock transfection with a DNA control 4 DNA quality Presence of contaminants Ensure that nucleic acid is pure contaminant free and endotoxin free Use high quality nucleic acids as impurities can lead to cell death 5 Concentration of transfection reagent nucleic acid too high Decrease the amount of nucleic acid reagent complexes added to the cells by lowering the nucleic acid amount or the transfection reagent concentration Complexes aggregation can cause some toxicity prepare them freshly and adjust
6. gt 90 of the activity of the reference lot 4 2 Troubleshooting Problems Comments and Suggestions Low 1 3D Fect nucleic acid ratio Optimize the reagent DNA ratio by using a fixed transfection amount of DNA ug and vary the amount of 3D Fect from 4 times less up to 1 5 times more efficiency than the suggested amount detailed in the Table 2 2 DNA amount Use different quantities of DNA with the recommended or optimized above 3D Fect DNA ratio 3 Cell density A non optimal cell density at the time of transfection can lead to insufficient uptake Optimal cell density is difficult to assess since a third dimension is added in cell culture try several densities depending on the support 4 DNA quality DNA should be as pure as possible and free of contaminants proteins phenol ethanol etc Endotoxins levels must be very low since they interfere with transfection efficiencies Employ nuclease free materials 5 Type of promoter Ensure that DNA promoter can be recognized by the cells to be transfected Other cells or viral driven reporter gene expression can be used as a control 6 Cell condition 1 Cells that have been in culture for a long time gt 8 weeks may become resistant to transfection Use freshly thawed cells that have been passaged at least once 2 Cells should be healthy and assayed during their exponential growing phase The presence of contaminants mycoplasma fungi alters
7. insert Completely biodegradable Universal primary cells and cell lines Multipurpose various types of nucleic acid Simple ready to use amp rapid Serum compatible Appropriate for multiple applications Long term transgene expression 2 Co SION Gl ss GS 1 2 Kit Content OZ Biosciences offers two sizes of 3D Fect transfection reagent e One tube containing 250 uL of 3D Fect good for 65 transfections with 1 ug of DNA e One tube containing 500 uL of 3D Fect good for 125 transfections with 1 ug of DNA e One tube containing 1 mL of 3D Fect good for 250 transfections with 1 ug of DNA Stability and Storage Storage Upon reception and for long term use store the reagent at 4 C for 3D Fect 3D Fect is stable for at least one year at 4 C Shipping condition Room temperature OZ Biosciences Protocol 3DFect vs 1 6 www ozbiosciences com 2 2 Applications O 2 1 Application Areas 3D Fect reagent has been developed for very efficient transfections of nucleic acids into a wide variety of immortalized and primary cells This transfection reagent is serum compatible and can be used for transient and stable transfection This product is stable ready to use and intended for research purpose only The field of applications covers tissue engineering tissue regeneration tumor invasion neural differentiation cellular polarization tissue formation colonization neurite growth antic
8. 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 3D Fect 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 3D Fect Reagent material and documentation to OZ Biosciences or by destroying all 3D Fect components Purchasers are advised to contact OZ Biosciences with the notification that a 3D Fect 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 3D Fect Reagent research only license and does not grant any other express or implied license The laws of the French Government shall govern the interpretation and enforcement of the terms of this Lic
9. ancer gene screening cell survival growth and differentiation co culture 2 2 Cell Types 3D Fect transfection reagent is suitable for numerous cells It has been successfully tested on a variety of immortalized and primary cells see results file An updated list of transfected cells is available on OZ Biosciences website www ozbiosciences com You can also submit your data to tech ozbiosciences com so we can update this list and give you all the support you need 3 General Protocols O 3 1 General Considerations The instructions given below represent sample protocols that were applied successfully to a variety of cells Optimal conditions vary depending on the nucleic acid cell types scaffolds types and cell culture conditions Therefore the amounts and ratio of the individual components DNA and 3D Fect may have to be adjusted to achieve best results Accordingly we suggest you to optimize the various transfection parameters as described in section 3 4 The following recommendations can be used as guidelines to quickly achieve very good transfection efficiency As a starting point we recommend to use 4 HL of 3D Fect Reagent 1 pg of DNA 3D Fect can be used in the presence or absence of serum You can use your routine culture medium for the transfection except during preparation of the 3D Fect DNA complexes see 3 3 below e Cells should be healthy and assayed during their exponential growi
10. considerably the transfection efficiency 7 3D matrix colonization Ensure that your cells have well colonized the 3D scaffold to promote or increase colonization we suggest to perform incubation of the cells and 3D matrix under slight agitation at 37 C 8 Medium used for preparing DNA 3D Fect complexes It is critical that serum free medium or buffer HBS PBS are used during the preparation of the complexes Avoid any direct contact of pure 3D Fect and DNA solutions with the plastic surface 9 Cell culture medium composition 1 For some cells transfection efficiency can be increased without serum or under reduced serum condition Thus transfect these cells in serum free medium during the first 12h of incubation 2 The presence of antibiotics might affect cell health and transfection efficiency 10 Incubation time and transfection volume The optimal time range between transfection and assay varies with cells promoter expression product etc The transfection efficiency can be monitored after 1 day Several reporter genes can be used to quantitatively monitored gene expression kinetics 11 Old 3D Fect DNA complexes The 3D Fect DNA complexes must be freshly prepared every time Complexes prepared and stored for longer than 1h can be aggregated 12 Transgene detection assay Ensure that your post transfection assay is properly set up OZ Biosciences Protocol 3DFect vs 1 6 www ozbiosciences com 7
11. e the cells at 37 C in a CO incubator under standard conditions until evaluation of the transgene expression Depending on the cell type and promoter activity the assay for the reporter gene can be performed from 1 to several days following transfection e For some cells 24h post transfection replace the old media with fresh media or just add fresh growth culture medium to the cells e In the case of cells very sensitive to transfection the medium can be changed immediately after cells have colonized the 3D Scaffold 3 4 Optimization Protocol Although high transfection efficiencies can be achieved in a broad range of cells and scaffolds with the rapid protocol optimal conditions may vary depending on the nucleic acid cell type 3D scaffold composition and complexity 3D scaffold volume and culture medium composition Therefore we recommend optimization of the protocol for each combination of plasmid cells and scaffold used in order to get the best out of 3D Fect reagent Consequently we suggest that you optimized these important parameters Ratio of 3D Fect to DNA The quantity of nucleic acid used The cell number Culture medium composition serum and reagent nucleic acid complex medium Incubation time We recommend that you optimize one parameter at a time while keeping the other parameters constant The two most critical variables are the ratio of 3D Fect reagent to DNA and the quantity of DNA OZ Biosciences
12. ense Product Use Limitations The 3D Fect 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 de Luminy Zone Luminy Entreprise 163 avenue de Luminy 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 business ozbiosciences com OZ Biosciences Protocol 3DFect vs 1 6 www ozbiosciences com 10
13. mL PI11000 Lipofection Technology lipid based Lullaby siRNA transfection reagent 1mL LL71000 DreamFect Gold Transfection reagent 1mL DG81000 DreamFect Transfection reagent 1mL DF41000 EcoTransfect Transfection Reagent 1mL ET11000 VeroFect Transfection Reagent 1mL VF61000 FlyFectin Transfection Reagent 1mL FF51000 CaPO Transfection Kit CP90000 Plasmids pVectOZ pVectOZ CAT 25yg PLOO010 pVectOZ GFP 25ug PLOO020 pVectOZ LacZ 25ug PLO0030 pVectOZ Luc 25yg PLO0040 pVectOZ SEAP 25ug PL00050 Gene amp Protein Tools Bradford Protein Assay Kit BA00100 GeneBlaster selection kit GB20010 GeneBlaster Emerald GB20014 B Galactosidase ONPG assay kits GO10001 B Galactosidase CPRG assay kits GC10002 X Gal Staining Kit GX10003 Biochemical D Luciferin Na 1g LN10000 D Luciferin K 1g LK10000 G 418 Sulfate 1g GS21000 X Gal powder 1g XG11000 Our dedicated and specialized technical support group will be pleased to answer any of your request and to assist you in your experiments Do not hesitate to contact us for all complementary information and remember to visit our website in order to stay inform on our last breakthrough technologies and updated on our complete product list http www ozbiosciences com OZ Biosciences Protocol 3DFect vs 1 6 www ozbiosciences com 9 Purchaser Notification Limited License The purchase of the 3D Fect Reagent grants the purchaser a non transferable non exclusive license to use
14. ng phase The presence of contaminants mycoplasma fungi will considerably affect the transfection efficiency The cell proliferating rate is a critical parameter and the optimal confluency has to be adjusted according to the cells used We recommend using regularly passaged cells for transfection and avoid employing cells that have been cultured for too long gt 2 months e Nucleic acids should be as pure as possible Endotoxins levels must be very low since they interfere with transfection efficiencies Moreover we suggest avoiding long incubation time of the DNA RNA solution in buffers or serum free medium before the addition of 3D Fect reagent to circumvent any degradation or surface adsorption e Antibiotics The exclusion of antibiotics from the media during transfection has been reported to enhance gene expression levels We did not observe a significant effect of the presence or absence of antibiotics with the 3D Fect reagent and this effect is cell type dependent and usually small e Materials We recommend using polypropylene tubes to prepare the DNA and transfection reagent solutions but glass or polystyrene tubes can also be used A protocol used for other transfection reagents should never be employed for 3D Fect and inversely Each transfection reagent has its own molecular structure biophysical properties and concentration which have an important influence on their biological activity 3 2 Cells Preparation It is
15. o be optimized 3 Cell number The cell proliferating rate is also a critical parameter and the optimal confluency has to be adjusted according to the cells used Thus the next step is to use the optimized ratio and DNA amount obtained previously and vary the cell number to be assayed 4 3D Fect DNA complex medium The buffer or medium composition use to prepare the 3D Fect DNA may influence the transfection efficiency For instance PBS can be used to prepare the DNA and 3D Fect solutions instead of serum free medium PBS composition 137MM NaCl 2 7mM KCI 1 5mM KH2PO and 6 5mM NazHPO x 2 H20 pH7 4 Other buffers such as HBS Tris can also be used 5 Effect of serum Transfection volume Almost all cell lines transfected with 3D Fect showed good results if serum is present during the transfection Some cell lines may behave differently and transfection efficiency can be increased without serum or under reduced serum condition Remember that presence of serum during complex formation must be avoided Transfection efficiency is delayed since cells have to attach and colonize 3D matrices before transfection can occur Consequently the cells may be kept in serum free or reduced serum conditions during the first 3 to 4 hours of transfection If you use serum free medium replace it by a culture medium containing serum or just add serum to the wells according to your standard culture condition after this period 6 Incubation time
16. or one hour at 37 C We recommend performing the scaffold re hydration under slight agitation 150 rpm For transfection experiments we advise transferring the hydrated sponge or scaffold to a suitable cell culture dish or well before adding the cells and then incubate under agitation for better colonization 1 Preparation of complexes Loading Cells on 3D Scaffold DNA Vol 50 or 100 pL 3D Fect 4uL per pg DNA Vol 50 or 100 pL 9 9 2 Incubation time 20 minutes at RT 6 Optionnal agitation 37 C gt 3 Complexes are added to 3D Scaffold 7 Assay 24 72H 4 1h agitation at 37 C a The DNA and 3D Fect reagent solutions should have an ambient temperature and be gently vortexed prior to use The rapid protocol is as simple as follows Use 4 uL of 3D Fect per ug of DNA We suggest beginning with this ratio and optimize it if required by following section 3 4 Important considerations before beginning transfection e 3D Fect reagent must be stored at 4 C e Do not use serum containing media for the preparation of DNA 3D Fect complexes step 1 OZ Biosciences Protocol 3DFect vs 1 6 www ozbiosciences com 4 1 Prevent the 3D Fect reagent and DNA stock solutions to come into contact with any plastic surface First add serum free culture medium to the tube and then drop the 3D Fect and DNA stock solution directly into the medium Contact of 3D Fect and DNA with the tube
17. recommended to seed the 3D Scaffolds on the day of transfection 3D scaffolds sponges The suitable cell density will depend on the growth rate the cells conditions and the size of the matrix In 3D cell culture the cell number can be increased in comparison to 2D systems For example the number of cells may vary from 10 000 cells to more than 100 000 cells for a 0 05 cm 3D Scaffold surface of 0 25 cm x 0 2cm height see results file OZ Biosciences Protocol 3DFect vs 1 6 www ozbiosciences com 3 The correct choice of optimal plating density also depends on the planned time between transfection and transgene analysis for a large interval prefer lower density and for a short interval a higher density may be advantageous see section 3 3 for procedure Optionally we suggest seeding cells on 3D scaffolds loaded with complexes under slight agitation 150 rpm from 4 to 24h to facilitate the matrix colonization Table 1 Cell number DNA amount 3D Fect volume and transfection conditions suggested for 3D Scaffolds Scaffold Size Adherent DNA 3D Fect Dilution Culture Cell Number ug Volume pL Volume pL Volume 0 05 cm 0 5 x 0 5 x 0 2 0 1 1x10 1 30r4 2x50 500 uL 0 125 cm 0 5x0 5x0 5 0 25 2x10 3 9 or 12 2x 50 500 HL 0 5 cm 1x 1x 0 5 1 10x10 15 45 or 60 2 x 100 1 mL 3 3 Protocol for 3D Scaffolds Before seeding the cells matrices must be hydrated with a solution of DNA mixed with 3D Fect reagent f
18. surface plastic or glass could result in materials lost by adsorption Preparation of DNA 3D Fect complexes 3D Fect DNA complexes are prepared in PBS or medium without serum because serum interferes with vector assembly 2 3 4 5 6 7 DNA solution Dilute 1 to 15 ug of DNA in 50 or 100 uL of PBS or culture medium without serum and antibiotics as indicated in Table 1 For optimization see section 3 4 3D Fect solution Allow the reagent to reach room temperature Dilute 3 to 60 uL of 3D Fect in 50 or 100 uL of PBS or culture medium without serum and antibiotics as indicated in Table 1 For optimization see section 3 4 Add the DNA solution into the 3D Fect solution mix gently by carefully pipetting up and down 2 3 times Do not vortex or centrifuge Incubate the mixture for 20 minutes at room temperature Place the 3D Scaffold in a suitable cell culture well or dish and add the complexes Try to avoid bubbles while hydrating the sponge it can be gently squeezed against the well wall to chase air bubbles Incubate the hydrated scaffold 1 hour at 37 C We recommend 150 rpm agitation for better complexes dispersion within the 3D Scaffold Transfer the hydrated 3D Scaffold into an appropriate well or dish and add cells see Table 1 in complete culture medium Option we suggest placing the cells under agitation 150 rpm at 37 C for 4 to 24h for a better scaffold colonization Incubat
19. the ratio as outlined previously 6 Incubation time Reduce the incubation time of complexes with the cells by replacing the transfection medium by fresh medium after 4h to 24h Our dedicated and specialized technical support group will be pleased to answer any of your requests and to help you with your transfection experiments tech ozbiosciences com In addition do not hesitate to visit our website www ozbiosciences com and the FAQ section OZ Biosciences Protocol 3DFect vs 1 6 www ozbiosciences com 8 5 Related Products Description Reference 3D Fection Technology 3D Fectin for all 3D hydrogels collagen hyaluronic acid PEG _TN30500 Magnetofection Technology Mega Magnetic Plate MF14000 Super Magnetic Plate MF10000 Magnetic Plate 96 magnets MF10096 PolyMag imL for all nucleic acids PN31000 PolyMag Neo imL for all nucleic acids PG61000 LipoMag Kit for all nucleic acids LM80500 CombiMag 1mL to boost transfection reagent CM21000 SilenceMag imL for siRNA application SM11000 NeuroMag imL for transfection of neurons NM51000 ViroMag imL for all viral applications VM41000 ViroMag R L 1mL for retrovirus and Lentivirus RL41000 AdenoMag 1mL for adenovirus AM71000 SelfMag Amino Kit SA10000 SelfMag Carboxy Kit SC20000 FluoMag P 100uL FP10100 FluoMag C 100uL FC10100 FluoMag S 100uL FS10100 FluoMag V 100uL FV10100 Protein Delivery Systems Ab DeliverIN 1 mL AI21000 Pro DeliverIN 1

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