liposome transfection

Summary

One of the cell transfection methods

Principle

Liposomes ((Iiposome) have been extensively studied as a vehicle for in vivo and in vitro delivery of carriers, and DNA wrapped in synthetic cationic lipids can be similarly brought into cells by fusion. The use of liposomes to bring DNA into different types of eukaryotic cells has been shown to have higher efficiency and better reproducibility than other methods.

Neutral liposomes utilize lipid membranes to encapsulate DNA, with the help of which the DNA is introduced into the cell membrane. With positively charged cationic liposomes, DNA is not pre-embedded in the liposomes, but the negatively charged DNA automatically binds to the positively charged liposomes to form a DNA-cationic liposome complex, which is then adsorbed onto the surface of the negatively charged cell membrane and introduced into the cell through endocytosis.

Appliance

cell transfection

Operation method

Liposome transfection

Materials and Instruments

Cells, Cell Culture Solution, Transfection Reagents, Petri dishes
EP Tubes, 6-Well Plates, Incubator

Move

(1) Cell culture: Take a 6-well culture plate (or a 35 mm dish), add 2 mL of culture medium containing ^1~2×105 cells to each well, and incubate in _CO2 at 37 ℃ until 40%~60% confluence (excessive confluence is detrimental to the screening of cells after transfection).

(2) Preparation of transfection solution: Prepare the following two liquids in EP tubes (the amount used for transfection of cells in each well).

Liquid A: Dilute 1~10 μg DNA with serum-free medium, total 100 μL;

Liquid B: Dilute 3 times the volume of transfection reagent with serum-free medium, totaling 100 μL, gently mix liquid A and B, and leave it at room temperature for 10~15 minutes, after which a slight turbidity phenomenon will appear, but it does not prevent transfection (if precipitation occurs, it may be due to too high a concentration of transfection reagent or DNA, and the amount should be reduced as appropriate).

(3) Preparation for transfection: Rinse twice with 2 mL of serum-free culture medium, then add 1 mL of serum-free culture medium.

(4) Transfection: add A/B complex slowly into the culture medium, shake well, and set in 37℃ for 6~24 hours, remove the serum-free transfection solution, and replace it with normal culture medium to continue the culture.

(5) The rest of the treatment, such as observation, screening, detection, etc., is the same as other transfection methods.

Caveat

Do not add serum during transfection; serum has a significant effect on transfection efficiency.

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