Product Focus — Polybrene, Transfection Enhancer

Product Introduction

Polybrene, also known as Hexadimethrine Bromide, is a polycationic polymer. Currently, Polybrene is widely used in retrovirus and lentivirus-mediated gene transfection. Its mechanism of action may be to promote adsorption by neutralizing the electrostatic repulsion between sialic acid on the cell surface and virus particles. Polybrene is also commonly used in DNA transfection experiments of mammalian cells to enhance the transfection efficiency of liposomes. It is also a well-known heparin antagonist, used in the production of non-specifically agglutinated red blood cells. In addition, since a small amount of Polybrene can increase polypeptide degradation in automatic sequencing analysis, Polybrene is also widely used in protein sequencing.

Usage Methods

This product is a sterile solution with a concentration of 10mg/mL. When used, it is generally diluted at a ratio of 1:1000-1:2000. The dilution ratio varies depending on the cell type. Please refer to relevant literature or adjust according to laboratory experience.

Experiment 1: Viral Infection

1.       Preparation of recombinant retrovirus stock solution: Add 5mL of growth medium to a 100mm culture dish containing approximately confluent transfected retroviral packaging cells. After incubating for 24h, aspirate the culture medium and filter it through a 0.45μm filter;

2.       Culture of cells to be infected: Add 10mL of complete medium to a 100mm culture dish with a cell density of approximately 5×10⁵/dish;

3.       Viral infection: After 24h of cell culture, aspirate the complete culture medium. Infect the cells with 2mL of viral supernatant containing Polybrene (or dilute the viral stock solution to 2mL) with a final concentration of Polybrene of 5μg-10μg/mL. Incubate at 37°C for 3-6h;

4.       Collection of virus particles: Add 8mL of complete medium. Three days after infection, lyse the cells with selective medium at a ratio of 1:5.

Experiment 2: DNA Transfection

1.       Culture cells in complete growth medium with a cell density of approximately 50%;

2.       After incubating the cells for 18-24h, prepare the DNA-medium-Polybrene mixture by following these steps:

      ✦ Add complete medium (2mL for 60mm culture dish, 3mL for 100mm culture dish) preheated at 37°C;

      ✦ Add 10ng~10µg of plasmid and mix gently;

      ✦ Add Polybrene to a final concentration of 5μg-10μg/mL. Mix gently. Each of the above components needs to be added in order.

3.       Remove the medium, add the DNA-medium-Polybrene solution to the cells, and incubate the cells at 37°C for 6-20h. Gently mix approximately every 1.5h within the first 6h of cell culture;

4.       Remove the DNA-medium-Polybrene solution. Gently cover the cells with DMSO shock solution (15% DMSO in 1× HBSS) (3mL for 60mm culture dish, 4mL for 100mm culture dish). Each time the solution is added, gently shake the culture dish by hand for 10s to distribute the liquid evenly. Then incubate the cells at 37°C for 4min;

5.       Immediately remove the DMSO shock solution and gently wash the cells twice with complete growth medium. For 60mm culture dishes, wash with 5mL of culture medium each time; for 100mm culture dishes, wash with 10mL of culture medium each time;

6.       Add complete medium to the cells;

7.       Stable transfection: Remove the growth medium and lyse the cells with selective medium at a ratio of 1:5. Transient expression: Remove the growth medium and add fresh growth medium. Harvest the cells after 24-72h.

Application Cases

Case 1: Polybrene Used in Lentivirus-Mediated Gene Transfection

In the experiment of screening polyclonal THP-1 cell lines stably expressing ORF3a, researchers added 8μg/mL Polybrene to infect THP-1 cells after collecting the lentivirus supernatant.

Case 2: Polybrene Used in Pseudovirus Neutralization Assay

In the study on the mechanism of ginkgolic acids inhibiting SARS-CoV-2 and its variants, the test compound was pre-incubated with hACE2/HEK293T cells at 37°C for 1h, then SARS-CoV-2 pseudovirus and 6μg/mL Polybrene were added to each well and incubated for 24h. IC50 was calculated 48h after infection.

    (Indicators: Percentage of Control, Pseudovirus Entry, NanoBiT Inhibition%; Statistical Symbol: p < 0.05, indicating significant difference; IC50 value is marked in the figure)

Reference: Ginkgolic acids inhibit SARS-CoV-2 and its variants by blocking the spike protein/ACE2 interplay. International Journal of Biological Macromolecules, 2023.

Case 3: Polybrene Used in Screening D-Phenotype Red Blood Cells

In the preliminary screening experiment, Polybrene was serially diluted at a 4/3 gradient to prepare gradient solutions. A mixed solution was prepared at a ratio of Polybrene gradient solution: IgG anti-D of 3:2. In a 96-well plate, 30μL of the above mixed solution was added to each well, then 0~35μL of LIM solution was sequentially added to each column (increasing by 5μL as a gradient), and finally 30μL of 3% red blood cell suspension was added, mixed well, centrifuged at 500×g for 5min, shaken at 750 rpm for 3min, and the results of different phenotype cells were observed.

Reference: Zhao F Y, Wang Z Y, Zhang Y Y, et al. Study on screening D-phenotype red blood cells by Polybrene [J]. Chinese Journal of Blood Transfusion, 2021.

Recommended Transfection-Related Products

Brand Mission

ANT BIO PTE. LTD. is dedicated to advancing life science research by providing high-quality, reliable reagents and comprehensive solutions. We recognize the critical challenges in gene transfection experiments and the urgent need for standardized, specialized experimental tools. Through our specialized sub-brands (Absin, Starter, UA), we have developed a targeted product portfolio for transfection research, covering core transfection enhancers (Polybrene), resistance screening reagents (G-418, Hygromycin B, etc.), various transfection reagents for different cell types, and supporting transfection media.

Our team adheres to stringent quality control standards throughout the product development and production process, ensuring the high purity, stability, and biological activity of each product. We are committed to providing professional technical support and customer-centric services, helping researchers overcome experimental challenges such as low transfection efficiency, difficult screening of stable cell lines, and poor adaptability of transfection reagents to different cell types, and accelerating the pace of scientific research breakthroughs in gene transfection-related fields. ANT BIO PTE. LTD. strives to be a trusted partner for scientists worldwide, contributing to the advancement of gene editing technology and the development of innovative biological research methods.

Disclaimer

This article is AI-compiled and interpreted based on the original work related to Polybrene (transfection enhancer) research. All intellectual property (e.g., experimental protocols, data, images) of the original publication shall belong to the relevant research team. For any infringement, please contact us promptly and we will take immediate action.

Brand Promotion Copy

ANT BIO PTE. LTD. – Empowering Scientific Breakthroughs

At ANTBIO, we are committed to advancing life science research through high-quality, reliable reagents and comprehensive solutions. Our specialized sub-brands (Absin, Starter, UA) cover a full spectrum of gene transfection needs, from core transfection enhancers and resistance screening reagents to specialized transfection reagents for different cell types and supporting media. With a focus on innovation, quality, and customer-centricity, we strive to be your trusted partner in overcoming gene transfection challenges and driving progress in gene editing technology. Explore our product portfolio today and elevate your research to new heights.