Cell transfection refers to a technique that introduces exogenous molecules into eukaryotic cells to alter their genotype or phenotype. With the continuous development of molecular biology and cell biology research, transfection has become a routine method for studying and controlling gene functions, widely applied in fields such as gene function research, regulation of gene expression, mutation analysis, as well as gene therapy, cell therapy, protein production, and vaccine production. Transfection can be roughly divided into three categories: physical-mediated, chemical-mediated, and biological-mediated approaches. Among them, chemical-mediated methods are widely used due to their advantages of high efficiency, low toxicity, convenience, and rapidity. Chemical-mediated methods include the classic calcium phosphate co-precipitation method, liposome transfection method, and various cationic substance-mediated transfection methods.
An ideal cell transfection method should have advantages such as high transfection efficiency and low cytotoxicity. Liposomes and cationic polymers are currently the most studied synthetic carriers. Liposomes have a high affinity for cell membranes; they can easily enter the cytoplasm through endocytosis or direct membrane fusion, and release the bound DNA through lipid-phospholipid exchange. Therefore, these materials usually have high transfection efficiency. However, liposomes have key limitations in gene delivery, including poor reproducibility in preparation and certain cytotoxicity. Many commercial liposome transfection reagents on the market require a high cell plating density, preferably 90%-95%, which helps reduce the impact of cytotoxicity caused by cationic liposomes. However, if the studied gene requires a relatively long expression time (e.g., cell cycle-related genes, cell surface proteins) or if subsequent culture and functional studies are needed after transfection, liposome nucleic acid transfection reagents are not suitable. Cationic polymer carriers are another type of widely studied synthetic carriers. These polymers form nano-sized complexes with DNA through ionic interactions. Cationic polymers have several unique characteristics in gene delivery, such as ease of manufacturing, stable formulation, and ease of modification. Currently, many researchers and biotech companies are focusing on the development of new-generation transfection reagents based on non-liposomal polymers to find more efficient and low-toxicity transfection reagents with less impact on research.
HP Nucleic Acid Transfection Reagent (H1505993) is a cationic polymer transfection reagent with superior performance, suitable for the transfection of DNA and RNA. Its principle is that positively charged polymers form positively charged complexes with the negatively charged phosphate groups of nucleic acids. These complexes then interact with negatively charged proteoglycans on the cell surface, enter the cell through endocytosis, and are subsequently released in the cytoplasm, achieving cellular transfection of exogenous nucleic acids. The cationic polymer used in HP Nucleic Acid Transfection Reagent (H1505993) has cell-penetrating peptides and endosome escape promoters introduced on its surface, which further enhance its endocytic capacity and endosome escape ability, ensuring that more complexes enter the cytoplasm and thus achieving better cell transfection effects.
HP Nucleic Acid Transfection Reagent (H1505993) has high transfection efficiency in various common cells, hard-to-transfect cells, and primary cells, with advantages such as high efficiency, low toxicity, simple operation, and good reproducibility. Its unique formula allows it to be directly added to the culture medium, and the presence of serum does not affect transfection efficiency, which can reduce cell damage caused by serum removal. Meanwhile, there is no need to remove the HP Nucleic Acid Transfection Reagent-nucleic acid complex or replace it with fresh medium after transfection, and the transfection system can also be optimized according to specific conditions.
Product Features:
⁕ Excellent transfection efficiency — It exhibits outstanding transfection efficiency across a wide range of cell types.
⁕ Low cytotoxicity — Transfected cells maintain good viability.
⁕ Broad application range — Comprehensive coverage of common cells, hard-to-transfect cells, and primary cells.
⁕ Simple operation — It maintains reliable transfection efficiency even in the presence of serum, and there is no need to remove the complex or replace with fresh medium after transfection.
Scope of Application:
HP Nucleic Acid Transfection Reagent uses advanced cationic polymer as its main component. It is a high-efficiency nucleic acid transfection reagent that can successfully transfect a variety of eukaryotic cells, hard-to-transfect cells, and primary cells, and is suitable for the transfection of DNA and RNA.
Summary of Experimental Procedure:
Figure 1. Flowchart of Transfection Experiment Using HP Nucleic Acid Transfection Reagent (H1505993)
Experimental Steps:
(Take a 24-well plate as an example; for the sample addition volume of other culture plates, refer to Table 1: Transfection Quantity Standards)
1. Prepare Cells for Transfection
(1) Adherent cells: One day before transfection, seed the trypsin-digested cells at a density of 0.5–1.5×10⁵ cells per well, ensuring the cell density reaches approximately 50% at the time of transfection.
(2) Suspension cells: On the day of transfection, before preparing the transfection reagent-nucleic acid complex, seed the cells in a 24-well plate by adding 1–3×10⁵ cells to 500 µL of growth medium per well.
⁕ Cell status significantly affects transfection efficiency. Cells to be transfected should be in a good growth state; it is recommended to use cells in the exponential growth phase with a survival rate >90% for transfection.
2. Prepare Transfection Reagent-Nucleic Acid Complex
(1) Add 0.5 μg of plasmid to a 1.5 mL centrifuge tube, then add 2 μL of transfection reagent to mix with the plasmid, and incubate at room temperature for 3 minutes.
⁕ The amount of transfection reagent used is affected by cell type and other experimental conditions. Under normal circumstances, the recommended DNA amount for a 24-well plate is 0.2–0.6 μg, and the recommended amount of transfection reagent is 1–4 μL. For first-time use, it is recommended to conduct pre-experiments with different dosages within the recommended range to optimize transfection efficiency.
(2) Add 100 μL of serum-free basal medium (Optimal-MEM Reduced Serum Medium (with Phenol Red) (Cat. No. O778393) is recommended) to the above mixture, mix gently, and let it stand at room temperature for 30 minutes to form the transfection reagent-nucleic acid complex.
⁕ The transfection reagent-nucleic acid complex remains stable at room temperature for up to 4 hours.
3. Cell Transfection
Replace the cell culture medium with fresh, pre-warmed complete medium (500 μL per well), then add the 100 µL transfection reagent-nucleic acid complex to each well of cells, and gently swirl the plate to mix.
⁕ For suspension cell lines: 5 hours after transfection, PMA and/or PHA can be optionally added to enhance the activity of the CMV promoter and promote gene expression. For Jurkat cells, the final concentrations of PHA and PMA are 1 µg/mL and 50 ng/mL, respectively, which can improve CMV promoter activity and gene expression. For K562 cells, adding PMA alone is sufficient to enhance promoter activity.
4. Analyze Transfected Cells
After culturing the transfected cells for 24–48 hours, the transfection efficiency can be detected by methods such as fluorescence detection, Western Blot, RT-PCR, ELISA, flow cytometry, or reporter gene assays according to actual needs. Alternatively, screening drugs can be added for the selection of stable cell lines.
Table 1: Transfection Volume Standards
Specifications of Cell Culture Equipment | Culture System (mL) | DNA (μg) | Transfection Reagent (μL) | Optimal-MEM Reduced Serum Medium (with Phenol Red) (O778393) |
96-well plate
| 0.1 | 0.1 | 0.4 | 20 |
24-well plate
| 0.5 | 0.5 | 2 | 100 |
12-well plate
| 1 | 1 | 4 | 200 |
6-well plate
| 2 | 2 | 8 | 400 |
60mm Dish
| 4 | 2-4 | 8-16 | 800 |
100mm Dish
| 10 | 5-10 | 20-40 | 2000 |
Precautions:
1. Nucleic Acid Quality: To achieve the highest transfection efficiency and lowest cytotoxicity, high-quality nucleic acids with high purity, sterility, no contamination, and no endotoxins should be used. Endotoxins in plasmids are a major obstacle to transfection, as they can significantly reduce transfection efficiency—especially for endotoxin-sensitive cells such as primary cells, suspension cells, and hematopoietic cells. It is recommended to use an endotoxin-free plasmid extraction kit for plasmid isolation, ensuring the plasmid has an A₂₆₀/A₂₈₀ ratio of 1.8–2.0. Meanwhile, the amount of plasmid used should be calculated appropriately; excessive plasmid transfection may cause cytotoxicity or even cell death.
2. Cell Quality: Cell status significantly affects transfection efficiency. It is recommended to use cells in the exponential growth phase with a survival rate >90% for transfection.
3. Cell Density: Transfection is recommended to be performed within 12–24 hours after cell passage, when the cell density is approximately 50%. Different cell transfection experiments have varying requirements for cell density. When transfecting different nucleic acids or different cell lines, experimental conditions need to be optimized according to the instruction manual. Additionally, consistent seeding conditions should be maintained throughout the experiment to ensure the reproducibility of experimental data.
4. Dosages of Plasmid and Transfection Reagent: For most cell lines cultured in 24-well plates, the recommended DNA dosage is 0.2–0.6 μg (0.5 μg is preferred), and the recommended transfection reagent dosage is 1–4 μL (2 μL is preferred). To obtain optimal transfection results, it is necessary to optimize the dosages of DNA and transfection reagent, and select appropriate dosages based on the type of transfected cells and plasmids.
5. HP Nucleic Acid Transfection Reagent can be used for transfection in serum-containing medium, and there is no need to remove the complex or replace the medium after transfection. However, when preparing the transfection complex, DNA and transfection reagent must be diluted in serum-free basal medium (Optimal-MEM Reduced Serum Medium (with Phenol Red) (Cat. No. O778393) is recommended), as serum can interfere with complex formation.
6. Since certain components in some special media may inhibit cationic polymer-mediated transfection, it is necessary to test the compatibility between the special medium and this product.
7. For your health and safety, please operate in a standardized manner and wear a lab coat and gloves during the experiment.
8. This product is for research use only and shall not be used for clinical diagnosis or treatment.
Experimental Case Analysis:
1. One day before transfection, the 9 types of cells in good condition (as shown in Figure 2) were seeded into a 24-well plate, ensuring the cell density reached approximately 50% at the time of transfection.
2. For each well, add 0.5 μg of GFP plasmid to a 1.5 mL centrifuge tube, then add 2 μL of transfection reagent (H1505993) to mix with the plasmid. After incubating at room temperature for 3 minutes, add 100 μL of Optimal-MEM Reduced Serum Medium (with Phenol Red) (Cat. No. O778393) to the mixture, mix gently, and let it stand at room temperature for 30 minutes to form the transfection reagent-nucleic acid complex.
3. Replace the cell culture medium with fresh, pre-warmed complete medium (500 μL per well), then add the 100 µL transfection reagent-nucleic acid complex to each well of cells, and gently swirl the plate to mix.
4. After culturing the transfected cells for 48 hours, the expression of GFP (Green Fluorescent Protein) was detected using a fluorescence microscope, and images were captured for documentation. The experimental results are shown in Figure 2.
Figure 2. GFP (Green Fluorescent Protein) expression was detected by fluorescence microscopy 48 hours after transfecting GFP plasmid (0.5 μg) using HP Nucleic Acid Transfection Reagent (H1505993).
