Technology

1.1. Description

MagnetofectionTM is a novel, simple and highly efficient method to transfect cells in culture. It exploits magnetic force exerted upon gene vectors associated with magnetic particles to draw the vectors towards, possibly even into, the target cells. In this manner, the full vector dose applied 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:

• Greatly improved transfection rates in terms of percentage of cells transfected compared to standard transfection.

• Up to several thousand fold increased levels of transgene expression compared to standard transfections upon short-term incubation.

• High transfection rates and transgene expression levels are achievable with extremely low vector doses, which allows to save expensive transfection reagents.

• Extremely short process time. A few minutes of incubation of cells with gene vectors are sufficient to generate high transfection efficiency, compared to several hours with standard procedures.

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response (expression level)

response (expression level)

Technology

1.4. Application

MagnetofectionTM is generally applicable for adherent cells and has been tested with a variety of immortalized cell lines and primary cells listed below. If a particular cell type or cell line is not listed this does not mean that MagnetofectionTM would not work. Also for the cells listed, some reagents have not been tested so far, as indicated by “n.d.” (not determined).

The CombiMAG reagent can be combined with any polycationic and lipidic transfection reagent, and also with adenoviral and retroviral vectors. In some cases, references are made in the footnotes to very successful combinations with commercially available reagents that have been tested so far.

Primary human umbilical vein endothelial cells positioned on the MagnetoFACTOR plate were incubated for 15 min with a Cy3 fluorescence- labeled antisense-oligonucleotide complexed with EffecteneTM (Qiagen; left) or EffecteneTM + CombiMAG (right).

(Data kindly provided by F. Kroetz. Ludwig-Maximilians University Munich)

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Technology

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Technology

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Technology

* Corresponds to successfully transfected by CombiMAG in association with different commercial transfection reagents such as FuGENETM (Roche), LipofectamineTM, METAFECTENETM (Biontex GmbH) and DMRIE-CTM (Invitrogen); adenovirus; EffecteneTM (Qiagen)

n.d. = not determined

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Utensils for MagnetofectionTM

2.1. MagnetoFACTOR plates

Apart from suitable magnetic nanoparticles, MagnetofectionTM requires appropriate magnetic fields. These are provided by the MagnetoFACTOR plate, especially designed for MagnetofectionTM.

The MagnetoFACTOR-96 plate 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 culture flasks, 6- and 12-well plates. In the larger plate formats, the MagnetoFACTOR plate will produce a pattern of higher and lower densities of transfected cells according to the geometry of the magnetic field lines. The MagnetoFACTOR-24 plate is special designed for the 24-well format.

The MagnetoFACTOR plates are compatible for well plates of the most manufacturer.

MagnetoFACTOR-96 plate MagnetoFACTOR-24 plate

SPECIAL OFFER

As an introductory offer you will receive one vial each of PolyMAG and CombiMAG sufficient for 200 μg of DNA each for free in combination with the purchase of the MagnetoFACTOR plate. This will enable you to optimize your MagnetofectionTM in up to 3000 transfection experiments (96-well format).

@ info@chemicell.com

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Avalaible Kits 3.1. Kits Contents

   9001
   9002
   9003

   9004
   9005
   9006

95 / 125 365 / 480 665 / 875

50 / 65 180 / 235 350 / 460

Product Number

9007

9009

9008-96

Description

PolyMAG-100

PolyMAG-500 PolyMAG-1000

CombiMAG-100

CombiMAG-500 CombiMAG-1000

MagnetoFACTOR-96 plate

MagnetoFACTOR-24 plate

SPECIAL OFFER:

MagnetoFACTOR-96 plate PolyMAG-200 CombiMAG-200

9008-24

Storage:

350 / 460

SPECIAL OFFER:

MagnetoFACTOR-24 plate PolyMAG-200 CombiMAG-200

Size

(sufficient for n μg DNA)

100

500 1000

100

500 1000

All components of the MagnetofectionTM kit should be stored at room temperatur (20-25°C). After first use store the kit at +4°C.

• Do not freeze the magnetic nanoparticles
• Do not add anything to the stock solution of magnetic nanoparticles • Shipping Conditions: Room Temperatur

–

–

–

200 200

–

200 200

Number of Transfection (96-well format)

1000

5000 10000

500 2500 5000

–

–

Price

EUR / USD

–

2000 1000

–

2000 1000

3.2. Contact

chemicell GmbH

Eresburgstrasse 22-23 12103 Berlin
Germany
Tel.: +49-30-2141481
Fax.: +49-30-21913737 e-mail: info@chemicell.com Internet: www.chemicell.com

@ info@chemicell.com

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12

Protocols

4.1. General Considerations

The instructions given below represent sample protocols that were applied successfully with a variety of cell lines. Please note that optimal conditions do vary from cell line to cell line. Therefore, the amount of DNA or RNA used and the ratios of the individual components may have to be adjusted to achieve best results. The following recommendations can be used as guidelines to achieve good transfection with minimal incubation times.

4.2. General Protocol

Adherent cells are seeded such that they reach 60-80% confluency at the time of MagnetofectionTM.

For suspension cells, use the specific protocol given in section 4.6. Immediately preceding transfection, the medium can be replaced with fresh medium (optionally without serum) if necessary.

The suggested cell number for adherent and suspension cells is given below.

Recommended DNA amount, PolyMAG volume and transfection volume.

*Total Transfection volume = culture medium + PolyMAG or CombiMAG complex

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Protocols

The following protocols (section 4.4, 4.5, 4.5.1, 4.5.2 and 4.6) can be used to produce stably transduced cells except that 48 hours post transfection. Cells are transferred to fresh medium containing the appropriate antibiotics for selection. It is important to wait at least 48 hours before exposing the transduced cells to selection media.

Vectors are prepared in medium without serum and supplement or in physiological saline, because serum may interfere with transfection complex assembly. The serum and supplement-free transfection complex is added to the cells that are covered with complete medium. Therefore, the addition of the transfection complex will result in the dilution of supplements such as serum, antibiotics or other additives of your standard culture medium. Although a medium changes after MagnetofectionTM is not required for most cell types, it may be necessary for cells that are sensitive to serum/supplement concentration. Alternatively, the cells may be kept in serum-free medium during MagnetofectionTM. In this case, a medium change will be required after MagnetofectionTM.

4.3. MagnetofectionTM in 6-, 12- , 24- or 96-well Plate Formats, T-75 Culture Flasks

As a rule of thumb, a DNA amount of 50 ng to 300 ng per square centimeter culture dish will yield good results. However, it is emphasized that every cell line requires optimization with respect to DNA amount and vector formulation.

The easiest way to generate the complexes is to provide the required amount of magnetic particles in a microcentrifuge tube, add the required amount of DNA which has been diluted with serum-free medium (e.g. DMEM). After 15 min incubation, add the magnetic particles / DNA complex to the cells. Position the 6-well culture plate on the MagnetoFACTOR plate for up to 10-20 min, subsequent perform a medium change (optional).

6-well culture dish:

A useful DNA amount for the 6-well format is 2 μg up to 6 μg, whereas the ratio of magnetic particles / DNA is 1:1. To 1.8 mL cells per well add 200 μL magnetic particles / DNA complexes.

12-well culture dish:

A useful DNA amount for the 12-well format is 2 μg up to 4 μg, whereas the ratio of magnetic particles / DNA is 1:1. To 0.8 mL cells per well add 200 μL magnetic particles / DNA complexes.

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Protocols 24-well culture dish:

A useful DNA amount for the 24-well format is 0.5 μg up to 2 μg, whereas the ratio of magnetic particles / DNA is 1:1. To 0.3 mL cells per well add 200 μL magnetic particles / DNA complexes.

96-well culture dish:

A useful DNA amount for the 96-well format is 0.1 μg up to 0.5 μg, whereas the ratio of magnetic particles / DNA is 1:1. To 0.15 mL cells per well add 50 μL magnetic particles / DNA complexes.

T-75 culture flask:

A useful DNA amount for the T-75 culture flask format is 15 μg up to 25 μg, whereas the ratio of magnetic particles / DNA is 1:1. To 14 mL (19 mL) cells add 1 mL magnetic particles / DNA complexes.

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Protocols

4.4. PolyMAG

Plate the adherent cells the day before transfection or suspension cells just before transfection in the appropriate tissue culture dish and volume of culture medium as recommended in table section 4.2.

The protocol is as simple as follows: Use 1 μL of PolyMAG per μg of DNA.

1. Add the required amount of PolyMAG (according to the DNA amount) to a microcentrifuge tube or to a U-bottom well of 96-well plate. If required and for amount less than 1 μL PolyMAG in your protocol, predilute PolyMAG with deionized water.

Note: Vortex the PolyMAG before used.
2. Dilute required amount of DNA to 200 μL with serum- and supplement-free

medium (such as DMEM).
3. Add the 200 μL DNA solution to PolyMAG and mix immediately by vigorous

pipetting.

4. After 15 minutes of incubation, add the 200 μL of complexes to the cells.

Note: The total transfection volumes per well (culture medium + PolyMAG complex) are suggested in the table above.

5. Place the cell culture plate upon the MagnetoFACTOR plate and incubate under standard cell conditions for 10 to 20 minutes.

6. Remove the MagnetoFACTOR plate.
6a.Optionally perform a medium change.
7. Cultivate cells under standard conditions until evaluation of transgene expression.

STANDARD TRANSFECTION MAGNETOFECTIONTM

Confluent primary human Keratinocytes transfected with PolyMAG
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Protocols

4.5. CombiMAG

A number of suppliers sell efficient transfection reagents. All of these can be made a magnetofectin by simple mixing with CombiMAG, usually resulting in strong improvements of these reagents efficiencies.

There are two strategies of using CombiMAG:

• One is to prepare a standard DNA complex with 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 mixing with the transfection reagent.

Also in this case, the instructions of the manufacturer are used with the only exception that instead of DNA alone, a mixture of DNA and CombiMAG is added to the transfection reagent.

Depending on the transfection reagent, the mixing order of components may influence the final transfection efficiency. It is recommended to use 1 – 2 μL of CombiMAG per μg 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.

Note: If required in your setup, predilute CombiMAG with ddwater.

CT-26 colon carcinoma cells were transfected for 15 min with a GFP reporter plasmid complexed with DMRIE-C (Invitrogen)

CT-26 colon carcinoma cells were transfected for 15 min with a GFP reporter plasmid complexed with DMRIE-C (Invitrogen) + CombiMAG on a magnetic plate

(Data kindly provided by Ch. Plank. Technical University Munich)

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Protocols

4.5.1 CombiMAG – Example Protocol for Fugene (Roche)

Plate 0.5 x 105 adherent cells per well the day before transfection or suspension cells just before transfection in a 24-well culture plate.

1.

2.

3.

4.

5.

6.

7.

Add 2.4 μL of Fugene to 97.6 μL serum- and supplement-free medium (such as DMEM) and vortex vigorously for 2 seconds.

Dilute 0.8 μg of DNA to 100 μL with serum- and supplement-free medium (such as DMEM).

Mix the DNA solution with the Fugene dilution by pipetting and incubate for 15 minutes at room temperature.

Add the resulting 200 μL of DNA complex to 1.6 μL of CombiMAG and mix immediately by vigorous pipetting. Vortex the CombiMAG before used.

After further 15 minutes of incubation add the DNA / Fugene / CombiMAG complex to the cells.

Place the cell culture plate upon the MagnetoFACTOR plate and incubate under standard cell culture conditions for 10 to 20 minutes.

Remove the magnetic plate and cultivate cells under standard conditions until evaluation of transgene expression.

8. Optionally perform a medium change.
9. Depending on the commercial transfection reagent used, this protocol may have

to be adapted.

Note: If required in your setup, predilute CombiMAG with ddwater.

Human Keratinocytes

Porcine Chondrocytes

Transfection of primary cells
Vector FugeneTM (Roche) +CombiMAG. 15 min transfection on MagnetoFACTOR plate (Data kindly provided by Ch. Plank. Technical University Munich)

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Protocols

4.5.2 CombiMAG – Example Protocols for other Cationic Lipid Reagents

The same steps as for Fugene are carried out. The primary DNA complex is prepared similar to the instructions of the manufacturer.

For example, in step 1, 3.2 μL of Lipofectamine + 96.8 μL medium or 4.0 μL GenePorter + 96 μL medium are used instead of Fugene. The rest of the protocol stays the same.

However, the user is reminded that the alternative mixing order (first mixing DNA and CombiMAG followed by mixing with lipid) may be advantageous.

Similarly, the polycationic reagents ExGen500 or Superfect (Qiagen) can be used instead of the lipids.

4.6. MagnetofectionTM – Suspension Cells

1. The composition and preparation of PolyMAG / DNA or CombiMAG / transfection

   reagent are performed exactly as described above (section 4.4 and 4.5).

2. While PolyMAG / DNA or CombiMAG / transfection reagent incubate for complex formulation, dilute the cells to be transfected to 5 x 105 – 1 x 106 / 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.

Option 1:

Option 2:

Option 3:

Seed the cells on polylysine-coated plates and use the protocol for adherent cells.

Briefly, centrifuge the cells (2 minutes) to pellet them and use the protocol for adherent cells.

Mix cell suspension with 30 μL of CombiMAG reagent per mL of cell suspension.

– Incubate for 10 – 15 minutes.

– 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 section 4.2.

– Incubate for 15 minutes

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Protocols

3. Add the resulting complex of PolyMAG / DNA or CombiMAG / transfection reagent to the cells while keeping the cell culture plate on the MagnetoFACTOR plate.

4. Continue to 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 MagnetoFACTOR plate.
   Be careful not to aspirate the magnetically sedimented cells.
6. Remove culture plate from MagnetoFACTOR plate.
7. Continue to cultivate cells as desired until evaluation of transgene expression.

4.7. MagnetofectionTM – siRNA

RNA interference is a powerful technique to shut down gene expression in cells and organisms. This silencing effect constitutes a very helpful tool to study gene function and is a promising approach for new therapeutic treatments. Short RNA duplexes (siRNA: small interfering RNA, shRNA: small hairpin RNA and dsRNA: double strand RNA) are extremely selective by interacting and inducing the degradation of their specific mRNA targets and thereby inhibit the resulting protein production.

PolyMAG introduces the siRNA duplexes in a variety of cells with a very high efficiency leading to exceptional knockdown effects with low doses of siRNA.

The protocol is very straightforward. Please refer to the tables below for specific amount of the respective compounds and transfection volume.

For instance use 2 μL of PolyMAG for 20 nM siRNA final concentration in a 6-well plate (transfection volume 2 mL)

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Protocols
Example dilution procedure of siRNA (1 μM stock solution*) and PolyMAG / CombiMAG :

*ng of siRNA was calculated on the basis of a MW = 13 500 g/mol

4.7.1 siRNA – PolyMAG Protocol

1. Plate the cells the day before transfection or just before transfection in your appropriate tissue culture dish and volume of culture medium as suggested (see table section 4.2).
2. Dilute the siRNA to 100 μL (or 200 μL) with culture medium without serum and supplement (such as DMEM) (see table for siRNA dilution procedure).
3. Vortex the PolyMAG tube before each use. If required, PolyMAG can be diluted only with deionized water.
   Don’t dilute PolyMAG with serum or supplement-free serum.
4. Add directly the appropriate volume/amount of PolyMAG to 100 μL (or 200 μL) of the diluted siRNA solution and mix immediately 4 – 5 times by vigorous pipetting.

5. Incubate the formed siRNA / PolyMAG complex 15 minutes at room temperature. 6. Add 100 μL (or 200 μL) of the binary complex drop by drop onto the cells.

Note: For some cells, serum-free condition for the first 3 hours of incubation might lead to better gene silencing. However, in most assays, siRNA delivery has been realized in culture medium with serum.

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Protocols
7. Place the cell culture plate upon the MagnetoFACTOR plate and incubate under

standard cell culture conditions for 10-20 minutes.

8. Remove the MagnetoFACTOR plate.

9. Cultivate the cells under standard conditions until evaluation of the gene silencing. Depending on the siRNA amount, the gene target and the cell type assays can be monitored 24 to 96 hours post-transfection.
We recommend 48 hours and 72 hours for RNA and protein knockdown analyses, respectively.

Note: Optionally a medium change can be performed 8-24 h after the transfection if your cells are sensitive to serum/supplement concentration.

4.7.2 siRNA – CombiMAG Protocol

For instance:

• Use 2 μL of CombiMAG for 20 nM siRNA final concentration in a 6-well plate (Transfection Volume 2 mL)

1.

2.

3.

Plate the cells the day before transfection or just before transfection in your appropriate tissue culture dish and volume of culture medium as suggested (see table section 4.2).

Prepare the binary siRNA / Cationic Lipid Reagent complex similar to the instructions of the manufacturer. The final volume should be 100 μl (or 200 μl) with culture medium without serum and supplement (such as DMEM) (see table for siRNA dilution procedure).

Vortex the CombiMAG tube before each use. If required, CombiMAG can be diluted only with deionized water.
Don’t dilute CombiMAG with serum or supplement-free serum.

4. Add directly the appropriate volume/amount of CombiMAG to 100 μL (or 200 μL) to the siRNA / Cationic Lipid Reagent complex and mix immediately 4 – 5 times by vigorous pipetting.

5. Incubate the formed siRNA / Cationic Lipid Reagent / CombiMAG complex 15 minutes at room temperature.

6. Add 100 μL (or 200 μL) of the ternary complex drop by drop onto the cells.

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Protocols

Note: For some cells, serum-free condition for the first 3 hours of incubation might lead to better gene silencing. However, in most assays, siRNA delivery has been realized in culture medium with serum.

7. Place the cell culture plate upon the MagnetoFACTOR plate and incubate under standard cell culture conditions for 10-20 minutes.
8. Remove the MagnetoFACTOR plate.
9. Cultivate the cells under standard conditions until evaluation of the gene silencing. Depending on the siRNA amount, the gene target and the cell type assays can be monitored 24 to 96 hours post-transfection. We recommend 48 hours and 72 hours for RNA and protein knockdown analyses, respectively.

   Note: Optionally a medium change can be performed 8-24 h after the transfection if your cells are sensitive to serum / supplement concentration.

Cell culture conditions:

Best results are achieved when cells are 60 – 80 % confluent at the time of the transfection. If necessary, you can wash the culture medium containing the transfection mixture after 8-24 hours and replace it by fresh medium.

siRNA concentration:

We often observed good siRNA effects by concentrations from 10 nM to 50 nM. However the efficiency may depend on the cell line, the target (half life, expression level…) and the siRNA used. Consequently, we suggest to start by testing a range of siRNA concentrations in order to obtain the best experimental conditions.

MagnetofectionTM Standard transection Untreated cells

Magnetofection of siRNA. Synthetic siRNA directed against eGFP was purchased from MWG Biotech, Ebersberg, Germany. The RNA was mixed with PEI- coated magnetic particles and additional free linear PEI (25 kDa, Polysciences, Warrington, PA, USA) in DMEM. After 30 min incubation, the mixture was diluted 51.2-fold with DMEM containing 10% FCS to result in a final siRNA concentration of 65 nM. Aliquots of this mixture (150 μL) were added to HT1080 cells (5000 cells/well in a 96-well plate) which had previously been stably transduced with a retroviral vector coding for eGFP. The culture plate was placed on a magnetic plate during the first 15 min of incubation. A medium change was performed after 24 h. The figure shows the cells 65 h after transfection, documenting an efficient knock down of eGFP expression.

(Data kindly provided by Ch. Plank. Technical University Munich)

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Protocols
4.8. MagnetofectionTM – Viruses – CombiMAG Protocol

Viral infection is highly cell surface receptor-dependent. For instance, adenoviruses are dependent on cells to express CAR (Coxsackie’s and adenovirus receptor) and HIV on cells to express CD4.

Unfortunately, many important and interesting target tissues for fundamental research and gene therapy are non-permissive to viral gene delivery (tumor tissues and apical surface of lung epithelium may express variable, little or none of the required receptors).

• The association of viral vectors with CombiMAG is sufficient to force infection of non-permissive cells as shown with adenovirus.

• MagnetofectionTM also increases retroviral infectious capacity.

NIH 3T3 cells (lacking the coxsackie and adenovirus receptor, CAR) were transfected with a recombinant adenovirus (coding for LacZ) mixed with CombiMAG in the presence and in the absence of permanent magnets positioned under the culture plates.

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Protocols

1. 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. Cells must be plated the day prior transfection.

2. Provide a suitable amount (see examples below) of CombiMAG in a tube large enough to contain the volume of virus preparation added in step 3.

3. Add your virus preparation (e.g. retroviral supernatant or purified adenovirus diluted in HBS, PBS or cell culture medium) to the tube(s) containing CombiMAG and mix immediately by pipetting or gentle vortexing. Thereafter, incubate 15 minutes at room temperature.

4. The ratios virus / CombiMAG should be adjusted according to the viral titers and cell types used. For optimization, we suggest as a starting point to use 1.5 μL,
3 μL, 6 μL and 12 μL of CombiMAG with a fixed quantity of virus preparation / supernatant.

5. Add the mixture prepared in step 3 to the cells in duplicate or triplicate.
6. Place the cell culture plate upon the magnetic plate and incubate under standard

cell culture conditions for 10-20 minutes.

7. Remove the MagnetoFACTOR plate. Optionally perform a medium change.

8. Cultivate the cells under standard conditions until evaluation of transgene expression.

9. Depending on the viral vector type, the quantity of virus and the cell types used, this protocol would have to be adjusted.

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Protocols

4.9. High Throughput Optimizations
The ratios of PolyMAG or CombiMAG to DNA can be varied by doubling or

multiplying the volumes of the reagents used.

Similarly, the reagents can be pre-diluted in deionized water and aliquots of the resulting dilutions are incubated with DNA or pre-formed DNA complexes, respectively, such as described above.

Finally, the assembled magnetofectins can be serially diluted to very low concentrations.

4.10. MagnetofectionTM – Optimization Protocol in 96-well Format

We recommend to optimize the transfection conditions in order to get the best results of MagnetofectionTM. Several parameters can be optimized:

• Ratio of PolyMAG / CombiMAG to nucleic acid or virus • Amount of nucleic acid
• Cell density
• Incubation time

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 μL medium per well.

1. In four tubes, dilute 7.2 μg DNA with 352.8 μL serum- and supplement-free medium (e.g. DMEM)

2. Add 3.6 μL, 7.2 μL, 10.8 μL and 14.4 μL of PolyMAG (in case of DNA) or CombiMAG (in case of DNA-transfection reagent complex) reagent in well A1, A4, A7 and A10 of a 96-well plate.

3. Add the 352.8 μL DNA solution from step 1 to well A1, A4, A7, A10 containing PolyMAG and mix well by pipetting. Incubate for 15 min at room temperature.

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Protocols

4. (Optional) Perform a medium change for the cells to be transfected. Remove medium the cells have been seeded in and replace with 150 μL fresh medium (with or without serum or supplements).
5. In the meantime add 180 μL serum- and supplement-free medium (e.g. DMEM) to the residual wells of column 1, 4, 7 and 10 of the 96-well plate (B1-H1, B4-H4, B7-H7, B10-H10).
6. After the incubation in step 3 transfer 180 μL from well A1, A4, A7, A10 to B1, B4, B7, B10, mix by pipetting, transfer 180 μL from B1, B4, B7, B10 to C1, C4, C7, C10, mix by pipetting and so on down to H1, H4, H7, H10.
7. Transfer 50 μL each in duplicate from column 1, 4, 7 and 10 to the columns of the cell culture plate where the cells to be transfected have been seeded
   (column 2/3 , 5/6 , 8/9 , 11/12).

8. Place the culture plate on the MagnetoFACTOR plate and incubate under cell culture conditions for 10-20 min.

9. Remove MagnetoFACTOR plate.
10. (Optional) Perform a medium change, particularly if the transfection has been

carried out in serum-free medium. 11. Continue to culture cells as desired.

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Protocols

4.11. Troubleshooting

Low Transfection Efficiency

• ▪  Inappropriate buffer composition:

  Serum-free buffer or medium has to be used for the formation of the PolyMAG/DNA- or CombiMAG/DNA complex, otherwise proteins from the serum will bind to PolyMAG or CombiMAG. Once the PolyMAG/DNA- or CombiMAG/DNA complex is formed it can be applied to cells in the presence of serum.

• ▪  Suboptimal ratio of PolyMAG or CombiMAG to nucleic acid or virus: Determine the optimal ratio of PolyMAG or CombiMAG to DNA by using the

  optimization protocol for 96-well plate (section 4.10).

• ▪  Correct handling of the MagnetoFACTOR plate:

  Use the MagnetoFACTOR plate with the magnets facing up. After addition of the PolyMAG/DNA- or CombiMAG/DNA complex to the cells, position the cell culture plate on the MagnetoFACTOR plate.

• ▪  Positive control:
  Perform a positive control transfection experiment with a well-characterized

  reporter gene (e,g. GFP, Luciferase).

• ▪  Mycoplasma contamination:
  Mycoplasma contamination alters transfection efficiency.
• ▪  Cell condition:
  Cells that have been in culture for a long time may become resistant to

  transfection. Use freshly thawed cells that have been passaged at least once.

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Protocols

Cellular Toxicity

• ▪  Cell density (% confluence) was not optimal at the time of transfection: Adherent cells are seeded such that they reach 60-80 % confluency at the time of MagnetofectionTM. If the cell density is too low, increased toxicity may be observed. For suspension cells it is necessary that the cells are immobilized on the well bottom (section 4.6).
• ▪  Suboptimal amount of DNA (section 4.10):
  Using MagnetofectionTM, approximately 5 x less DNA compared to Lipofection is

  necessary.

• ▪  Purity of transfecting molecule:
  Check the purity of the molecule of interest to be delivered (lipopolysaccharides

  which are endotoxins will cause cell death).

• ▪  Amounts of PolyMAG or CombiMAG:
  Higher amounts of PolyMAG or CombiMAG may be cell toxic and can additional

  reduce the transfection efficiency.

• ▪  Incubation time on the MagnetoFACTOR plate:
  Longer incubation times (e.g. 4 hours) with PolyMAG/DNA- or CombiMAG/DNA

  complex plus application of an magnetic field can cause toxic effects.

• ▪  Incubation time after MagnetofectionTM:
  Reduce the incubation time of complexes with the cells. Transfection medium can

  be replaced by fresh medium after 4 hours.

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References

1. 9.001  Plank C., Scherer F., Schillinger U., Anton M. Magnetofection: Enhancement and localization of gene delivery with magnetic particles under influence of a magnetic fields. J. Gene Med. 2000; 2((5) Suppl.): S24.
2. 9.002  Scherer F., Anton M., Schillinger U., Henke J., Bergemann C., Krüger A., Gänsbacher B. and Plank C. Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo. Gene Therapy 2002; 9: 102-109.
3. 9.003  Plank C., Schillinger U., Scherer F., Bergemann C., Remy J.-S., Krötz F., Anton M., Lausier J. and Rosenecker J. The Magnetofection Method: Using Magnetic Force to Enhance Gene Delivery. Biol. Chem. May 2003; 384: 737-747.
4. 9.004  Plank C., Anton M., Rudolph C., Rosenecker J. and Krötz F. Enhancing and targeting nucleic acid delivery by magnetic force. Expert Opinion on Biological Therapy 2003; 3: 745-758.
5. 9.005  Plank C., Scherer F., Schillinger U., Bergemann C., Anton M. Magnetofection: enhancing and targeting gene delivery with superparamagnetic nanoparticles and magnetic fields. J. Liposome Res. 2003; 13(1): 29-32.
6. 9.006  Plank C. Magnetofection: enhancing and targeting gene delivery with lipid-DNA vectors by magnetic force. J. Liposome Res. 2003; 13(1): 105-106.
7. 9.007  Krötz F., Son HY., Gloe T., Planck C. Magnetofection Potentiates Gene Delivery to Cultured Endothelial. Journal of Vascular Research 2003; 40: 425-434.
8. 9.008  Krötz F., Wit C., Sohn HY., Zahler S., Gloe T., Pohl U. and Plank C. Magnetofection-A highly efficient tool for antisense oligonucleotide delivery in vitro and in vivo. Mol. Ther. May 2003; 7(5): 700-710.
9. 9.009  Bridell H. Magnetic field guided nucleic acid delivery to target the Raf-1 kinase in human umbilical vein endothelial cells. Master thesis in medical science 2003, Technical University of Munich, Germany.
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