Cat.-no	Description	Amount	Price €	Shop
30250	3D-Plus Transfection Reagent Maximo	250 µl	119.00	add
30500	3D-Plus Transfection Reagent Maximo	500 µl	209.00	add
31000	3D-Plus Transfection Reagent Maximo	1 ml	399.00	add

3D-Plus Transfection reagent: Manual


3D Transfection: a novel perspective for your cells !

3D-Plus Transfect reagent is the newest reagent specifically developed to directly transfect cells cultured in 3D hydrogel. 3D-Plus Transfect reagentis suitable for all kind of hydrogels and cells.

3D matrices not only add a third dimension to cells’ environment, they also allow creating significant differences in cellular characteristics and behavior. Because 3D matrices are routinely used in basic research and therapeutic applications, we have developed 3D-Plus Transfect reagenttransfection reagent specific for hydrogel. In this way, hydrogel-based 3D matrices combined with 3D-FectIN™/DNA complexes, are colonized by cells to be transfected in a more natural environment. This method allows studying angiogenesis, tube and acini formation, colonization, neurite growth, tissue engineering, tissue regeneration, tumor invasion, neural differentiation, cellular polarization, tissue formation…

3D-Plus Transfect reagentis the newest reagent specifically developed to directly transfect cells cultured in 3D hydrogel.

This reagent is based on a novel technology that allows adding a third dimension to cell culture.

• Highly efficient: on cell lines and primary cells
• Specific for 3D hydrogels
• All type of nucleic acids: plasmid DNA, siRNA, shRNA...
• Completely biodegradable
• Serum compatible
• Long term protein expression

 High transfection efficiency in various cells

3D-Plus Transfect reagenthas been successfully tested on a variety of cells. Transfection efficiency and transgene expression achieved are high and long-lasting.
Transfection of various cells on different gels




High secreted alkaline phosphatase expression




Efficient with various type of hydrogels and matrices

Name	Type of Scaffold
Collagen	Collagen-based hydrogels
Collagen-derived	Collagen-derived hydrogels
HA	Hyaluronic acid
Gelatin	Extracellular Matrix (ECM)
Fibrin / Fibronectin	ECM
Fibrinogen	ECM
Laminin	ECM
Matrigel™ *	BD Bioscience
Poly-(Ethylene Glycol)	PEGylated hydrogels

Outperforms competitors
Comparison of RFP expression in HEK-293 cells




Comparison of secreted alkaline phosphatase production in NIH-3T3 cells



Trouble shooting:

Problems

	Comments and Suggestions
Low transfection efficiency                                       Low transfection efficiency	1- 3D-PLUS Transfect / nucleic acid ratio. Optimize the reagent / DNA ratio by using a fixed amount of DNA (µg) and vary the amount of 3D-PLUS Transfect from 2 times less up to 2.5 times more than the suggested amount detailed in the Table 2.  2- Hydrogel / Dilution rate. The protocol is design for a 2X dilution of gel. Try reducing the volume of DNA/3D-PLUS Transfect mix to change final gel dilution.  3- DNA amount. Use different quantity of DNA with the recommended or optimized (above) 3D-PLUS Transfect  / DNA ratio.  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- DNA quality. Nucleic acids 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.  6- 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.  7- Cell condition. 1) Cells that have been in culture for a long time (> 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 considerably the transfection efficiency.  8- Medium used for preparing DNA / 3D-PLUS Transfect  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-PLUS Transfect and pure DNA solution 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-PLUS Transfect  / DNA complexes. The 3D-PLUS Transfect  / 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 and includes a positive control.  13- 3D-PLUS Transfect 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-PLUS Transfect is kept more than one week at +4°C. Store at -20°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.  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 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 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.

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