Protocol for Enhanced Transfection of Mammalian Cells with Polybrene

In Brief

Polybrene (Cat. No. 7711) is a cationic polymer that improves lentiviral and adenoviral transduction efficiency in mammalian cells in vitro. It also enhances DNA transfection in many types of cells. It has a neutralizing effect on negatively charged DNA and viral particles which facilitates their attachment and entry into host cells. Polybrene is suitable for use with CRISPR/Cas9 protocols. It is commonly used in combination with Protamine sulfate (Cat. No. 8822) to enhance the delivery of adeno-associated virus (AAV) and lentiviral vectors.

Polybrene is a lyophilized solid and can be prepared as a stock solution by adding 1 mL of sterile ultra-pure water directly to the vial (final stock concentration 10 mg/mL).

Step 1: Amplify and Isolate High-Quality Plasmid DNA

Sufficient plasmid DNA must first be generated. Begin by amplifying the quantity of the plasmid by performing bacterial transformation. Grow the transformed bacterial culture overnight (in 37°C shaker) and extract the plasmid DNA using DNA MAXIprep kit.

Step 2: Culture HEK293T Cells to Optimal Confluency

Culture HEK293T cells in HEK media (DMEM/F-12 media, FBS, non-essential amino acids, L-glutamine) within humidified incubators (standard conditions of 37˚C, 5% CO₂ and 21% O₂). Once the culture has reached high confluency (>90%), the cells are ready for transfection.

Step 3: Prepare Transfection Media with Polybrene and Lipofection Reagents

• Immediately before transfection, replace the HEK culture media with DMEM.

• Prepare the transfection media, which consists of an optimized ratio of DMEM, DNA plasmid, lipofection reagent and polybrene stock solution (see Table 1). Typically, polybrene is used at a concentration of 4 µg/mL - 10 µg/mL (see Table 2).

   

Step 4: Perform Polybrene-Enhanced Transfection

• Gently mix the transfection media before adding dropwise to the culture plate, to prevent damaging the cells.

• Place the cells immediately in the incubator with the following settings overnight: 35˚C, 3% CO₂ and 21% O_2.

   

Step 5: Post-Transfection Incubation and Sample Harvesting

• Return culture to standard maintenance incubator. Leave the cells for 48-72 h post-transfection before proceeding to downstream application (i.e., harvesting of virus from media).

Polybrene Stock Solution Preparation (Table 1)

Polybrene (Cat. No. 7711) is supplied as a 10 mg solid and is soluble in water (up to 100 mg/mL). To generate a working solution, Polybrene should first be dissolved in 1 mL sterile water to generate a 10 mg/mL stock solution.

For more information on preparing stock solutions, see our Molarity Calculator.

Polybrene Working Solution Preparation (Table 2)

Polybrene working solution is generated from the polybrene stock solution (10 mg/mL in sterile water). This stock solution can be further diluted in the culture media to generate the desired working concentration for the transfection or transduction media. Example dilutions shown below.

For more information on calculating dilutions see our Dilution Calculator.

Frequently Asked Questions

• How to troubleshoot low transfection efficiency when using Polybrene?

  Check plasmid quality, Polybrene concentration, timing of media changes, cell confluency, and incubation conditions. 

• How should stock solutions be stored?

  Polybrene stock solutions should be aliquoted and stored at -20°C to avoid repeated freeze-thaw cycles, which can degrade Polybrene's activity.

• How important is the plasmid DNA purity for successful transfection?

  Highly pure, endotoxin-free plasmid DNA at the correct concentration is critical to maximize transfection efficiency.

• Why are lower CO₂ and temperature settings (3% CO₂, 35°C) used during transfection?

  Reduced CO2 and temperature decrease cellular metabolic stress and improve transfection efficiency by stabilizing DNA-lipid complexes and enhancing cellular uptake.