昆虫细胞转染试剂是一款能够有效地将bacmid质粒转入Sf9、Sf21和HighFive™等昆虫细胞的转染试剂。该产品适用于Bac-to-Bac®、BaculoDirect™和InsectSelect™表达系统的蛋白表达，具有操作简单、表达量高、细胞毒性低等优势。产品以无菌的液体形式提供。
实验材料：
• 本产品包含昆虫细胞转染试剂
• 高纯度的核酸储液（0.5～5μg/μL）
• 无血清无抗生素昆虫培养液
质量控制：
• 4°C保存条件下有效期一年
• 本产品经严格的质量检验证明，无生物污染
注意事项：
• 常温下制备昆虫细胞转染试剂-核酸复合物，孵育时间共25min。转染时使用不含抗生素和血清的培养基和灭酶管。
• 利用本产品转染孔板中的细胞4-6h后，去除原培养基，更换为新鲜含血清培养基；利用本产品转染摇瓶培养的悬浮细胞时，无需更换培养基。
• 本产品应该在4°C密闭保存，避免反复冻融。
• 初次使用应该优化核酸浓度与昆虫细胞转染试剂的比例以得到最高的转染效率。
• 转染时请使用适当保存和经常传代的昆虫细胞，并确保培养基无细菌、真菌或支原体污染，如细胞是近期复苏的液氮冻存细胞，请在转染前至少传代两次。
• 转染试剂使用后请立即盖好盖子，避免长时间暴露在空气中，影响转染效率。
转染效果示例：
图1. 昆虫细胞转染试剂和国产竞品在Sf9细胞中递送bacmid质粒96h和120h后的明场照片和荧光场照片
图2. 昆虫细胞转染试剂和国产竞品在Sf9细胞中递送bacmid质粒120h后的流式结果图
按照下表转染细胞。每种反应混合物体积为单个孔的体积，且考虑了移液差异。按比例计算其他孔的体积。
昆虫细胞转染试剂----转染步骤详细说明（以6孔板为例）
1.用正常的细胞培养基，按照每孔8×10⁵个细胞，接种细胞至70-90%的密度，在27-28°C培养15min。
2.铺板15min后，将孔板内的正常细胞培养基换为800μL无血清无抗生素昆虫培养液。
3.将3.2μgDNA加入100μL无血清无抗生素昆虫培养液中，得到DNA稀释液。
4.将40μL昆虫细胞转染试剂加入100μL无血清无抗生素昆虫培养液中，并静置5min（昆虫细胞转染试剂的用量可在30-45μL范围内进行优化，以寻求最佳转染效果）。
5.将步骤1中的DNA稀释液与步骤2中的昆虫细胞转染试剂稀释液充分混合均匀，室温下孵育20min。
6.将孵育好的昆虫细胞转染试剂–DNA复合物加至细胞。
7.27°C孵育细胞4-6h后，将培养基更换为2mL新鲜的完全培养基，继续27°C孵育细胞5-6天后分析转染细胞。
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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