si3d-fectin INSTRUCTION MANUAL 3D Transfection Reagent dedicated to sirna delivery into Hydrogels: Collagen, Hyaluronic acid, PEG, Fibrin, Laminin

Transcription

1 si3d-fectin INSTRUCTION MANUAL 3D Transfection Reagent dedicated to sirna delivery into Hydrogels: Collagen, Hyaluronic acid, PEG, Fibrin, Laminin

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4 si3d-fectin Instruction Manual si3d-fectin transfection reagent 3D Transfection: a novel perspective for your cell culture!! si3d-fectin is the latest OZ Biosciences reagent for 3D transfection of sirna in hydrogels. It has been specifically designed and developed for gene silencing of cells cultured in 3D hydrogels (collagen, hyaluronic acid, PEG, laminin ). This reagent is based on a novel technology that allows adding a third dimension to cell cultures. List of si3d-fectin Kits Catalog Number Description Volume (µl) Number of transfections / 50 nm sirna in 50 µl Gel STN50250 si3d-fectin STN50500 si3d-fectin STN51000 si3d-fectin 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, ) or directly through our website. For all other supplementary information, do not hesitate to contact our dedicated technical support: tech@ozbiosciences.com. OZ Biosciences SAS 163 avenue de Luminy Case 922, zone entreprise Marseille cedex 09 - FRANCE Ph: +33 (0) Fax: +33 (0) contact@ozbiosciences.com order@ozbiosciences.com OZ Biosciences INC 4901 Morena Blvd, Suite 501 San Diego CA USA Ph : Fax : contactusa@ozbiosciences.com orderusa@ozbiosciences.com -1-

5 Table of Contents 1. Technology Description Kit Contents 3 2. Applications 3 3. General Protocols General Considerations Cells Preparation Protocol for Hydrogels Optimization Protocol 6 4. Appendix Quality Controls Troubleshooting Related Products 9 6. Purchaser Notification Technology 1.1. Description si3d-fectin is the newest transfection reagent specifically designed and developed for silencing gene expression in cells cultured in gels (or hydrogels). 3D matrices not only add a third dimension to the cells environment, they also allow creating significant differences in cellular phenotype and behavior. Because 3D environments are routinely used in basic research and for therapeutic applications, OZ Biosciences has continued developing reagents for 3D applications. Two new powerful reagents, si3d-fectin (for hydrogels) and si3d-fect (for scaffolds) are now dedicated to gene silencing. In this way, 3D matrices bearing complexes formed with si3d-fectin reagent and sirna are colonized by cells to be transfected in a more natural environment. si3d-fectin reagent associated with 3D matrices allows numerous cell transfections in order to study angiogenesis, tube and acini formation, colonization, neurite growth, tissue engineering, tissue regeneration, tumor invasion, neural differentiation, cellular polarization, tissue formation Principal si3d-fectin advantages: 1. Highly efficient for gene silencing in 3D 2. Ideal for any Gel and Hydrogel 3. Dedicated to short nucleic acid sequences (sirna, mirna ) 4. Completely biodegradable 5. Universal (primary cells and cell lines) 6. Simple, ready-to-use & rapid 7. Serum compatible 8. Long term gene silencing -2-

6 1.2. Kit Content OZ Biosciences offers three sizes of si3d-fectin transfection reagent. One tube of 250 µl of si3d-fectin good for transfections using 50 nm sirna in 50 µl gel. One tube of 500 µl of si3d-fectin good for 65 to 125 transfections using 50 nm sirna in 50 µl gel. One tube of 1 ml of si3d-fectin good for 125 to 250 transfections using 50 nm sirna in 50 µl gel. Stability and Storage Storage: Upon reception, store the reagent at 4 C si3d-fectin is stable for at least one year at +4 C. Shipping condition: Room Temperature. 2. Applications si3d-fectin reagent has been developed for very efficient transfection of sirna into a wide variety of immortalized and primary cells. This transfection reagent is serum compatible and can be used for highly efficient gene silencing. This product is stable, ready-to-use and intended for research purpose only. The field of applications covers angiogenesis, tube and acini formation, colonization, neurite growth, tissue engineering, tissue regeneration, tumor invasion, neural differentiation, wound healing, cellular polarization, tissue formation An updated list of successfully transfected cells is available on OZ Biosciences website: 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 3.1. General Considerations The instructions given below represent sample protocols that were applied successfully to a variety of cells. Optimal conditions may vary depending on the nucleic acid, cell types, hydrogel types and cell culture conditions. Therefore, the amounts and ratio of the individual components (sirna and si3d-fectin ) may have to be adjusted to achieve best results. As a starting point, use 4 to 8 µl of si3d-fectin Reagent with 50 nm final sirna in 50 µl gel. Cells should be healthy and assayed during their exponential growing phase. The presence of contaminants (mycoplasma, fungi) will considerably affect the transfection efficiency. The optimal confluency has to be optimized according to the cells and the culture dish used. We recommend using regularly passaged cells for transfection and avoid employing cells that have been cultured for too long (> 2 months). Short nucleic acids should be as pure as possible, free of DNA, proteins and other contaminants. Endotoxins levels must be very low since they interfere with transfection efficiencies. We suggest avoiding long storage of the diluted nucleic acid solution before the addition of si3d-fectin to circumvent any degradation or surface adsorption. Culture medium. 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 si3d-fectin transfection reagent. -3-

7 3.2. Cells Preparation It is recommended to seed the hydrogels on the day of transfection. The suitable cell density will depend on the growth rate, size, ability to invade hydrogels and the cells conditions. Moreover each hydrogel bears specific characteristics regarding the cell type to be used. In 3D cell culture, the cell number can be increased in comparison to 2D systems; please refer to Table 1 below for recommended cell culture conditions. The optimal plating density also depends on the planned time between transfection and gene silencing analysis: for a large interval, prefer lower density as gene silencing generally occurs later than gene expression. Table 1: Cell number, si3d-fectin volumes and transfection conditions suggested for 50 nm final sirna in 50 µl gel. Total Hydrogel Volume Adherent Cell Number si3d-fectin Volume (µl) Dilution Volume (µl) Hydrogel volume * (µl) 50 µl x to 8 2 x µl x to 16 2 x µl 1 4 x to 32 2 x *Generally, gels must be diluted 50/50 volume with sirna/si3d-fectin complexes. A 50% dilution should be compatible with polymerization capacities of gels and should not interfere with cell growth. Before transfection experiments, we recommend testing cell culture on a 50% gel diluted with medium. Otherwise, we suggest reducing the dilution volume for the preparation of the sirna/si3d- FectIN complexes Protocol for Hydrogels Important considerations before beginning transfection: si3d-fectin reagent must be stored at +4 C. Do not use serum-containing media for the preparation of sirna/si3d-fectin complexes (step 1)! Prevent the si3d-fectin reagent and sirna stock solutions to come into contact with any plastic surface. First, add serum-free culture medium to the tube and then drop the si3d-fectin and sirna stock solution directly into the medium. Contact of si3d-fectin and sirna with the tube surface (plastic or glass) could result in material lost by adsorption. For thermo-sensitive gels (i.e. Matrigel TM*, BD Biosciences) work on ice with 4 C cooled pipet tips for mixing complexes and gels (step 3) in order to keep gel in its liquid, non-polymerized form for better complexes dispersion. -4-

8 The sirna and si3d-fectin solutions should be at room temperature and gently vortexed prior to use. The rapid protocol is as simple as follows: Use 4 to 8 µl of si3d-fectin with 50 nm of sirna in 50 µl gel. We suggest beginning with these volumes (4, 6 and 8 µl) and optimize conditions by following section ) Reagents preparation. Allow reagent to reach room temperature before beginning. a. sirna solution. Dilute sirna in 12.5 to 50 µl of PBS of culture medium without any supplement (SVF, antibiotics, growth factors ) for a final concentration of 50 nm. b. si3d-fectin solution. Briefly vortex the reagent before use. Dilute 4 to 32 µl of si3d-fectin in 12.5 to 50 µl of PBS of culture medium without any supplement (SVF, antibiotics, growth factors ); refer to table 1. For optimization see section ) Complexes formation. Combine the sirna and the si3d-fectin solutions. Mix gently by carefully pipetting up and down and incubate the mixture for 20 minutes at room temperature. Do not vortex or centrifuge! Note: Proceed to step 3 within 30 minutes 3) Mix the complexes with 25 to 100 µl of hydrogel. Avoid bubbles while dispersing complexes within the hydrogel. This step is crucial since complexes dispersion should be done rapidly (to keep the hydrogel in its liquid state) while avoiding bubbles that could interfere with transfection. 4) Dispatch these complexes-containing hydrogel in suitable culture dish or well and incubate at 37 C for 30 minutes for gel polymerization. 5) Add cells in complete medium on the gel. Gently rock the plate to homogenize cell suspension. 6) Let the cells colonize the hydrogel and incubate them at 37 C in a CO 2 incubator under standard conditions until evaluation of the gene silencing. Depending on the cell type and sirna design, the assay for the gene silencing can be performed from 1 to several days following transfection. 4-5-

9 Note: For some cells, 24h post-transfection replace the old media with fresh media or just add fresh growth culture medium to the cells. Note: In the case of cells very sensitive to transfection, the medium can be changed immediately after cells have colonized the gel Optimization Protocol To achieve the highest transfection efficiency, the following parameters can be optimized: Ratio of si3d-fectin to sirna: Maintain a fixed quantity of sirna (according to the size of your gel or cell number) and then vary the amount of si3d-fectin reagent over the suggested range in the Table 2 below. Table 2: Suggested range of si3d-fectin for optimization using 50 nm sirna. Total Hydrogel Volume si3d-fectin Volume (µl) si3d-fect volume (µl) proposed interval 50 µl µl µl The quantity of nucleic acid used Once the optimal si3d-fectin / sirna ratio is found, adjust the sirna concentration according to Table 3. Table 3: Suggested range of sirna amounts for optimization with si3d-fectin. Total Hydrogel Volume sirna (nm) sirna concentration (nm) proposed interval 50 µl 100 µl 200 µl Finally, culture medium composition (for preparing the complexes), cell density, total culture medium volume and incubation time can also be optimized. We recommend you to optimize one parameter at a time while keeping the other parameters constant. The two most critical variables are the ratio of si3d-fectin reagent to sirna and the concentration of sirna. -6-

10 4. Appendix Our dedicated and specialized technical support team will be pleased to answer any of your requests at In addition, do not hesitate to visit our website Quality Controls To assure the performance of each lot of si3d-fectin produced, we qualify each component using rigorous standards. The following in vitro assays qualify the function, quality and activity of each component. Specification Standard Quality Controls Sterility Thioglycolate assay. Absence of fungal and bacterial contamination shall be obtained for 7 days. Biological Activity Gene silencing efficiency on COS-7_GFP and NIH-3T3_GFP cells. Every lot shall have an acceptance specification of > 90% of the activity of the reference lot Troubleshooting Problems Low transfection efficiency Comments and Suggestions 1- Optimization of si3d-fectin / nucleic acid ratio. See section sirna amount. Use different quantities of sirna with the ratio. 3- Hydrogel / Dilution rate. The protocol is designed for a 2X dilution of gel. Try reducing the volume of sirna/si3d-fectin mix to change final gel dilution. 4- 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. 5- sirna quality. sirna should be as pure as possible and free of contaminants (proteins, phenol, ethanol, salt, etc.). Endotoxins levels must be very low since they interfere with transfection efficiencies. Employ nuclease-free materials. 6- Cell condition. 1) Cells cultured for a long time (> 8 weeks) may become resistant to transfection. Use freshly thawed cells that have been passaged at least once. 2) The presence of contaminants (mycoplasma, fungi) alters considerably the transfection efficiency. 7-3D matrix colonization. Ensure that your cells have well colonized the hydrogel. 8- Medium used for preparing sirna / si3d-fectin complexes. It is critical that serumfree medium or buffer (HBS, PBS) are used during the preparation of the complexes. 9- Cell culture medium composition. 1) For some cells, transfection efficiency can be increased in absence of serum. Transfect these cells in serum-free medium during the first 4h. 2) The presence of antibiotics might affect cell health and transfection efficiency. 10- Incubation time. The optimal time range between transfection and gene silencing varies with cells, sirna target, etc. The transfection efficiency can be monitored after 1 day. 11- Old si3d-fectin / sirna complexes. The si3d-fectin / sirna complexes must be freshly prepared every time to avoid aggregation. 12- Gene Silencing assay. Ensure to use a validated sirna sequence; try using a pool of sirna to guaranty the gene silencing. 13- si3d-fectin reagent temperature. Reagent should have an ambient temperature. 14- Transfection reagent storage. Transfection efficiency can slowly decrease if si3d- FectIN is kept more than one week at RT. Store at 4 C to recover initial efficiency. -7-

11 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) Check the cell density, 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 gel will allow cell to attach or not; non adhered cells can undergo apoptosis. 3- Key Gene silencing. If the targeted gene is essential for cell survival or if a key-gene is non-specifically silenced by the sirna this can lead to cell death. Use suitable controls such as transfection reagent alone or mock transfection with a control or scrambled sirna. 4- sirna quality - Presence of contaminants. Ensure that nucleic acid is pure, contaminantfree and endotoxin-free; 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 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. * Matrigel TM is a trademark own by BD Biosciences. -8-

12 5. Related Products Description 3D TRANSFECTION TECHNOLOGY 3DfectIN (for hydrogels culture) 3Dfect (for scaffolds culture) MAGNETOFECTION TECHNOLOGY Transfection reagents: PolyMag Neo (for all nucleic acids) Magnetofectamine kit: Lipofectamine CombiMag (for all nucleic acids) NeuroMag (dedicated for neurons) SilenceMag (for sirna application) Transfection enhancer: CombiMag (to improve any transfection reagent efficiency) Viral Transduction enhancers: ViroMag (to optimize viral transduction) ViroMag R/L (specific for Retrovirus and Lentivirus) AdenoMag (for Adenoviruses) In vivo Magnetofection In vivo ViroMag (for magnetic assisted viral infection) In vivo PolyMag (polymer-based magnetic nanoparticles) In vivo DogtorMag (lipid-based magnetic nanoparticles) LIPOFECTION TECHNOLOGY (LIPID-BASED) Lullaby (sirna transfection reagent) DreamFect Gold (Transfection reagent for all types of nucleic acids) DreamFect Stem (Transfection reagent for Stem Cells) RmesFect and RmesFect Stem (Transfection reagent for mrna) PROTEIN DELIVERY SYSTEMS Ab-DeliverIN (delivery reagent for antibodies) Pro-DeliverIN (delivery reagent for protein in vivo and in vitro) PLASMIDS PVECTOZ pvectoz-lacz / pvectoz-seap / pvectoz-gfp / pvectoz-luciferase ASSAY KITS Bradford Protein Assay Kit β-galactosidase assay kits (CPRG/ONPG) MTT cell proliferation kit BIOCHEMICALS G-418, Sulfate 1g X-Gal powder 1g D-Luciferin, K+ and Na+ 1g 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

13 Purchaser Notification Limited License The purchase of the si3d-fectin Reagent 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. 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 si3d-fectin 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 si3d-fectin Reagent material and documentation to OZ Biosciences, or by destroying all si3d-fectin components. Purchasers are advised to contact OZ Biosciences with the notification that a si3d-fectin 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 si3d-fectin 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 License. Product Use Limitations The si3d-fectin 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 SAS Parc Scientifique de Luminy Zone Luminy Entreprise 163, avenue de Luminy Case Marseille Cedex 9 - FRANCE Ph: +33 (0) Fax: +33 (0) business@ozbiosciences.com -10-

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16 CONTACTS OZ Biosciences SAS 163 avenue de Luminy Case 922, zone entreprise Marseille cedex 09 FRANCE Ph: +33 (0) Fax: +33 (0) OZ Biosciences INC 4901 Morena Blvd, Suite 501 San Diego CA USA Ph : Fax : contactusa@ozbiosciences.com orderusa@ozbiosciences.com techusa@ozbiosciences.com Follow us!