Precautions ï¬ It is recommended to dilute Lipofectamine 2000 and nucleic acids using Opti-MEM I low serum medium (Cat. No. 31985-062) prior to mixing. ï¬ Do not add antibiotics to the medium during transfection to avoid cell death. ï¬ Please keep the cell inoculation conditions the same for different batches of experiments. ï¬ Please check the compatibility of serum-free medium with Lipofectamine 2000, as some serum-free media (such as CD293, SFM II, VP-SFM, etc.) may inhibit cationic liposome-mediated transfection. Transfection with liposome RNAi or siRNA The following steps are applicable to mammalian cells cultured in 24-well plates. For other culture materials, please refer to the transfection scale adjustment. All quantities and volumes are calculated per well. 1. On the day before transfection, cells were seeded in 500 μl of antibiotic-free medium to achieve 30-50% confluency during transfection. 2. Preparation of transfection solution, the amount of cells per well is as follows: A. Dilute 20 pmol siRNA (33 nM final siRNA concentration) with 50 μl Opti-ME I low serum medium (or other serum-free medium) and mix gently. B. Shake Lipofectamine 2000 gently before use, then dilute 1 μl of Lipofectamine 2000 in 50 μl Opti-ME I medium and incubate for 5 minutes at room temperature. Note: Please proceed to the next step within 25 minutes. C. Mix the DNA diluted in the first two steps with Lipofectamine 2000 (to make the total volume 100 μl), mix gently, and let stand at room temperature for 20 minutes (the solution may appear turbid). 3. Add 100 μl of transfection solution to each well of the cells and shake gently. Gene expression was detected after 24-96 hours of incubation at 37 ° C, and the medium was changed after 4-6 hours of transfection. Optimization of liposome siRNA transfection The amount of siRNA and Lipofectamine 2000 can be adjusted to optimize transfection. In 24-well plates, siRNA can be adjusted between 10-50 pmol, Lipofectamine 2000 can be adjusted between 0.5-1.5 μl, and optimization should be optimized when increasing cell density. Dyeing dose. For more RNAi transfection techniques, please refer to "Successful RNAi Seven Steps" Liposome DNA transfection The following steps are applicable to mammalian cells cultured in 24-well plates. For other culture materials, please refer to the transfection scale adjustment. All quantities and volumes are calculated by well. Most cell lines use a ratio of DNA (μg) to Lipofectamin 2000 (μl) of 1:2 to 1:3, and transfection of high-density cells results in high transfection efficiency, high expression levels, and low cytotoxicity. Optimized transfection is required (see Optimizing Lipid DNA Transfection). 1. Adherent cells: 0.5-2×105 cells per well inoculated in 500 μl of antibiotic-free medium on the day before transfection, the cells can grow to 90-95% confluence during transfection. Suspension cells: 4-8 x 105 cells per well were seeded in 500 μl of antibiotic-free medium before formulating the transfection solution. 2. Preparation of transfection solution, the amount of cells per well is as follows: A. Dilute the plasmid DNA with 50 μl Opti-ME I low serum medium (or other serum-free medium) and mix gently. B. Shake Lipofectamine 2000 gently before use, then dilute in 50 μl Opti-ME I medium with appropriate amount of Lipofectamine 2000 and incubate for 5 minutes at room temperature. Note: Please proceed to the next step within 25 minutes. C. Mix the DNA diluted in the first two steps with Lipofectamine 2000 (to make the total volume 100 μl), mix gently, and let stand at room temperature for 20 minutes (the solution may appear cloudy). Note: Transfection complexes remain stable for 6 hours at room temperature. 3. Add 100 μl of transfection solution to each well of the cells and shake gently. 4. The gene expression was detected after 18-48 hours of incubation at 37 ° C, and the medium was changed after 4-6 hours of transfection. 5. For stable transfection, inoculate in fresh medium at a 1:10 dilution 24 hours after transfection and add the selection medium the next day. Optimized liposome DNA transfection Transfection can be optimized by varying cell density, plasmid DNA density, and Lipofectamine 2000 concentration. To ensure cell fusion over 90%, DNA (μg): Lipofectamin 2000 (μl) can be adjusted from 1:0.5 to 1:5. Transfection scale adjustment The amount of the different culture material transfection solution, cells and medium is converted according to the area of ​​the culture material. In a 96-well plate cell high-throughput automated analysis system, it is recommended to use 50 μl of transfection solution per well. Note: In 96-well plates, cells can be directly inoculated into the transfection solution for rapid transfection. 100 μl of the transfection solution is prepared directly in the culture plate, and the cells are directly added to the culture plate at a density twice that of the above method. The cells can adhere normally in the transfection solution. Features: Lipofectamine 2000 transfection of eukaryotic cells has the following advantages: High transfection efficiency for a variety of cells and culture vessels, successfully transfected cell lines The nucleic acid-Lipofectamine 2000 complex (transfection solution) can be added directly to the cell culture medium, whether or not the medium contains serum. ï¬ It is not necessary to remove the transfection solution after transfection, or change or add the medium, but the transfection solution can be removed after 4-6 hours of transfection. Active Pharmaceutical Ingredients Active Pharmaceutical Ingredients(API) refer to the raw materials used in the production of various preparations. They are the effective ingredients in the preparations. They are various powders, crystals, extracts, etc., prepared by chemical synthesis, plant extraction or biotechnology, but Substances that the patient cannot take directly. API is intended to be used in any substance or mixture of substances in the manufacture of pharmaceuticals, and when used in pharmaceuticals, it becomes an active ingredient of the pharmaceuticals. Such substances have pharmacological activity or other direct effects in the diagnosis, treatment, symptom relief, treatment or prevention of diseases, or can affect the function or structure of the body. According to its source, active pharmaceutical ingredients are divided into two categories: synthetic chemical active Pharmaceutical ingredients and natural chemical active Pharmaceutical ingredients. Chromium Picolinate,Tianeptine,6-Paradol,Aminobutyric acid,acetylcysteine,L-Carnosine Xi'an Gawen Biotechnology Co., Ltd , https://www.ahualyn-bios.com