The Insect Cell Transfection Reagent is a transfection reagent that can effectively deliver bacmid plasmids into insect cells such as Sf9, Sf21, and HighFive™. This product is suitable for protein expression in Bac-to-Bac®, BaculoDirect™, and InsectSelect™ expression systems, and has advantages including simple operation, high expression level, and low cytotoxicity. It is supplied as a sterile liquid.
Experimental Materials:
• This product includes the Insect Cell Transfection Reagent.
• High-purity nucleic acid stock solution (0.5–5 μg/μL).
• Serum-free and antibiotic-free insect cell culture medium.
Quality Control:
• The shelf life is 1 year when stored at 4 °C.
• Strict quality inspection has confirmed that this product is free from biological contamination.
Precautions:
• Prepare the Insect Cell Transfection Reagent-nucleic acid complex at room temperature, with a total incubation time of 25 minutes. For transfection, use antibiotic-free and serum-free medium as well as enzyme-free tubes.
• After transfecting cells in well plates with this product for 4-6 hours, remove the original medium and replace it with fresh serum-containing medium; when transfecting suspension cells cultured in shake flasks with this product, there is no need to replace the medium.
• This product should be stored tightly sealed at 4°C, and repeated freezing and thawing should be avoided.
• For initial use, the ratio of nucleic acid concentration to the Insect Cell Transfection Reagent should be optimized to achieve the highest transfection efficiency.
• During transfection, use insect cells that are properly preserved and regularly passaged, and ensure that the medium is free of bacterial, fungal, or mycoplasma contamination. If the cells are recently revived from liquid nitrogen frozen storage, they should be passaged at least twice before transfection.
• After using the transfection reagent, please cap it immediately to avoid prolonged exposure to air, which may affect transfection efficiency.
Example of transfection effect:
Figure 1. Bright-field images and fluorescence-field images of bacmid plasmid delivered by the Insect Cell Transfection Reagent and domestic competing products in Sf9 cells at 96h and 120h.
Figure 2. Flow cytometry results of bacmid plasmid delivered by the Insect Cell Transfection Reagent and domestic competing products in Sf9 cells at 120h.
Transfect cells according to the table below. The volume of each reaction mixture refers to the volume for a single well, with pipetting differences taken into account. Calculate the volumes for other wells in proportion.
Insect Cell Transfection Reagent----Detailed Instructions for Transfection Steps (Taking 24-well Plate as an Example)
1. Using normal cell culture medium, inoculate cells at a density of 70–90% with 8×10⁵ cells per well, and culture them at 27–28°C for 15 minutes.
2. Fifteen minutes after plating, replace the normal cell culture medium in the well plate with 800 μL of serum-free and antibiotic-free insect cell culture medium.
3. Add 3.2 μg of DNA to 100 μL of serum-free and antibiotic-free insect cell culture medium to prepare the DNA dilution.
4. Add 40 μL of Insect Cell Transfection Reagent to 100 μL of serum-free and antibiotic-free insect cell culture medium, and let it stand for 5 minutes (The dosage of Insect Cell Transfection Reagent can be optimized within the range of 30–45 μL to achieve the best transfection effect).
5. Thoroughly mix the DNA dilution from Step 3 with the Insect Cell Transfection Reagent dilution from Step 4, and incubate the mixture at room temperature for 20 minutes.
6. Add the incubated Insect Cell Transfection Reagent–DNA complex to the cells.
7. After incubating the cells at 27°C for 4–6 hours, replace the medium with 2 mL of fresh complete medium. Continue incubating the cells at 27°C for 5–6 days, then analyze the transfected cells.
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