Transfection Reagent Protocol

Mammalian Transfection Protocol for 96 well plates

Our reagent is based upon the delivery of synthetic surface ligands to the cell to induce stronger and more efficient surface binding of the assembled lipoplex which contains receptors specific the delivered synthetic cell ligands. This allows for the reduction of cationic charge of the lipoplex resulting in lower toxicity and higher transfection rates of a wider range of cell lines which are resistant to other transfection methods.

Transfection

Contents

Vial A – Blue cap (To Engineering Cell Surfaces with Bio-orthogonal Groups)

Vial B – Green cap (To Generate Nucleic Acid Complex)

General Considerations

Resulting successful transfections involves optimizing the application of Surface Engineering Liposomes, Chemolipoplex Reagent :DNA ratio and total amount of DNA used to transfect the target cells.
Surface Engineering liposomes: The application of VIAL A solution (5%-10% by volume of cell media) is a pretreatment to make the cells more susceptible to uptake our lipoplex (VIAL B+pDNA). Although these pretreatment liposomes (VIAL A) possess little to no toxicity to most cell lines, the over application of this solution results in greater toxicity due to increased lipids in the cell membrane and greater uptake of lipoplexes. The recommended dose of (VIAL A) is 5% over 5-10 minutes for easily/moderately transfectable cells but if the cell line is highly resistant to pDNA transfection then 1-4 hours incubation with (VIAL A) with no more then 5% vol/vol can be used without increasing cytotoxicity.
Not all transfections require the use of VIAL A. This reagent can be potentially omitted from specific protocols, since it is used to increase efficiency but if efficiency is already high for a particular cell line VIAL A application can be omitted to stream line transfection operations.
Some of the below protocols will confer different advantages to transfection depending upon the cell line of interest.

1.1 Protocol 1 – Traditional Serum Free Transfection

This protocol can be used to optimally transfect cells lines which are not sensitive to the absence of serum containing media

Cells are initially seeded unto their preferred substrate 16-24 hours before transfection and incubated using their preferred growth medium under standard conditions with a confluency between 60-90% depending upon their rate of growth.

1.2 Preparation of Transfection Reagent

Beforehand the Chemolipoplex must be generated by aliquoting VIAL B (5µL/per well) into a microtube followed by the addition of pDNA (0.1µg/per well) (pDNA concentration range between 1.4 - 0.14µg/µL is acceptable) and gently agitated with the pipette and allowed to incubate for 30 minutes at room temperature. Once the incubation period is complete the solution is diluted to 10% of the initial volume using serum free media with no antibiotics added e.g. to 20µL of chemolipoplex 200µL of serum free media is added.

1.3 Preparation of the Adherent Cells

The initial growth media is removed and then replaced with fresh serum containing media, to the fresh media add between 5-10% by volume of VIAL A solution (The serum containing growth media and VIAL A can be premixed before the addition to adherent cells). Use pipette agitation to thoroughly mix VIAL A solution and serum containing media and allow to incubate for 5 minutes (for increased results with difficult to transfect cell lines allow incubation with VIAL A solution (5% only) between 1-4 hours for increased transfection efficiency). The pretreatment is then drained and the cells are gently washed with PBS and subsequently drained and 100µL of serum free media is added.

Once the plated cells have media added the chemolipoplex solution can be added, between 5-10µL per well (7µL recommended starting point). The well is then gently pipetted to mix the solution thoroughly for the side of the well to prevent displacing the cells from the substrate. The serum free media can then be replaced 4-6 hours later with serum containing media if required. The cells are then incubated for 24-48 hours under standard conditions followed by checking for protein expression.

2.1 Protocol 2 – Traditional Transfection in the Presence of Serum

This protocol can be used to transfect cell lines which are sensitive to the absence of serum containing media.

2.2 Preparation of Transfection Reagent

Beforehand the Chemolipoplex must be generated by aliquoting VIAL B (7µL/per well) into a microtube followed by the addition of pDNA (0.2µg/per well) (pDNA concentration range between 1.4 - 0.14µg/µL is acceptable) and gently agitated with the pipette and allowed to incubate for 30 minutes at room temperature. Once the incubation period is complete the solution is diluted to 10% of the initial concentration using serum free media with no antibiotics added e.g. to 20µL of chemolipoplex 200µL of serum free media is added.

2.3 Preparation of Cells

Once the plated cells have media added the chemolipoplex solution can be added, between 5-10µL per well (7µL recommended starting point). The well is then gently pipetted to mix the solution thoroughly for the side of the well to prevent displacing the cells from the substrate. The cells are then incubated for 24-48 hours under standard conditions followed by checking for protein expression.

3.1 Protocol 3 – Direct SwitchOut Protocol

This protocol can be used to transfect cell lines which are sensitive to the absence of serum containing media, while are difficult to transfect in the presence of serum (secondary protocol).

3.2 Preparation of Transfection Reagent

Beforehand the Chemolipoplex must be generated by aliquoting VIAL B (5µL/per well) into a microtube followed by the addition of pDNA (0.1µg/per well) (pDNA concentration range between 1.4 - 0.14µg/µL is acceptable) and gently agitated with the pipette and allowed to incubate for 30 minutes at room temperature. Once the incubation period is complete the solution is diluted to 10% of the initial concentration using serum free media with no antibiotics added e.g. to 20µL of chemolipoplex 200µL of serum free media is added. From this stock solution the chemolipoplex may require further dilution to 1/5 to 1/10 concentration i.e. 5µL to 25µL or 5µL to 50µL respectively.

3.3 Preparation of Cells

The initial growth media is removed and then replaced with fresh serum containing media, to the fresh media add between 5-10% by volume of VIAL A solution (The serum containing growth media and VIAL A can be premixed before the addition to adherent cells). Use pipette agitation to thoroughly mix VIAL A solution and serum containing media and allow to incubate for 5 minutes (for increased results with difficult to transfect cell lines allow incubation with VIAL A solution (5% only) between 1-4 hours for increased transfection efficiency). The pretreatment is then aspirated and the cells are gently washed with PBS, aspirated, followed by the immediate addition of the prepared chemolipoplex reagent solution i.e. the chemolipoplex should be added to the cells just after aspiration of the PBS wash.

As a recommended starting point individual 96 wells should treated with 10µL of diluted chemolipoplex and incubated for 5- 10 minutes before the addition of serum containing media without aspiration. If the cell line is resistant to transfection this incubation period can be extended and is dependant upon the cells sensitivity to serum free conditions. The cells are then incubated under standard conditions for 24-48 hours and checked for protein expression.

4.1 Protocol 4 – Transfection of Suspended/Non-adherent Cells

This protocol can be used to transfect cell lines which are non-adherent or required to be quickly transfected upon removal from storage and possibly sensitive to the absence of serum containing media

Transfection of non-adherent or suspended cells in media solution can be transfected using this reagent by following standard cell thawing protocols or (plating) protocols with minimal added cell manipulation steps.

4.2 Preparation of Transfection Reagent

Beforehand the Chemolipoplex must be generated by aliquoting VIAL B (7µL/per well) into a microtube followed by the addition of pDNA (0.1µg/per 100µL of final cell suspension volume) (pDNA concentration range between 1.4 - 0.14µg/µL is acceptable) and gently agitated with the pipette and allowed to incubate for 30 minutes at room temperature. Once the incubation period is complete the solution is diluted to 10% of the initial concentration using serum free media with no antibiotics added e.g. to 20µL of chemolipoplex 200µL of serum free media is added.

4.3 Preparation of Non-Adherent Cells

For spinning culture of non-adherent cell lines the initial growth media is removed through centrifugation and then replaced with fresh serum containing media, to the fresh media add between 5-10% by volume of VIAL A solution (The serum containing growth media and VIAL A can be premixed before the addition the cell suspension). Use agitation or spinning to thoroughly mix VIAL A solution, serum containing media and cells, followed by 5 minutes of incubation for 5-10 minutes (for increased results with difficult to transfect cell lines allow incubation with VIAL A solution (5% only) between 1-4 hours for increased transfection efficiency). The VIAL A treated on-adherent cells are then centrifuged again and the media is aspirated and the cells are gently washed with PBS and subsequently centrifuged and aspirated followed by the addition of serum containing media as required for normal cell maintenance.

To transfect the VIAL A treated cell suspension Before addition to the cells, the lipoplex solution is diluted using PBS or serum free growth media and can then either be added directly to drained cell plates or added to cells already treated with serum free media.

4.4 Preparation of Suspended Adherent Cells

The initial growth media is aspirated and the cells are washed with PBS according to standard cell culture cell expansion techniques ie. Trypsinization. Once the cells have been removed from the plate and are in suspension at a suitable cell concentration e.g. 1X10⁶-6X10⁶ cells/mL through the addition of either fresh serum containing media or serum free media, add between 5-10% by volume of VIAL A solution (The serum containing growth media and VIAL A can be premixed before the addition to adherent cells). Use pipette agitation to thoroughly mix VIAL A solution and serum containing media and allow to incubate with the cells for 5 minutes (for increased results with difficult to transfect cell lines allow incubation with VIAL A solution (5% only) between 1-4 hours for increased transfection efficiency). The cells can be plated without removal of the VIAL A treatment solution until they become affixed to the tissue culture substrate and then aspirated and washed once with PBS before chemolipoplex treatment but the cells can recentrifuged, aspirated and the pellet resuspended for plating onto tissue culture plates and immediately treated with the chemolipoplex.

Once the cells are suspended in the appropriate serum containing media the chemolipoplex solution can be added. The chemolipoplex solution should be added with a ratio of 100µL per 1mL of suspended cell solution as a recommended starting point. The solution should be well mixed using gentle pipetting. The cells are then incubated for 24-48 hours under standard conditions followed by checking for protein expression.