Liposome-mediated transient transfection
Liposome-mediated transient transfection
This lab was derived from: Animal Cell Culture - A Guide to Basic Techniques (5th Edition)
Operation method
Scheme 27.12 Liposome-mediated transient transfection
Materials and Instruments
Target cell DNA Move A . Transfection of adherent cells For more product details, please visit Aladdin Scientific website.
Cationic lipids D-PBSA buffered salt solution NaCl 0.25% trypsin
Cell culture medium Reduced serum medium Serum-free medium Multi-well plates
1. Inoculate 1. 3X105 cells/well into a 6-well culture plate with 3 ml of medium.
2. Incubate at 37°C in CO2 incubator until 50%~90% confluence. This process takes at least 16 h, usually 18-24 h. The cells are then incubated at 37°C in CO2 incubator until 50%-90% confluence.
3. Before transfection, prepare DNA and liposome solutions in sterile test tubes:
(a) For DNA solution, dilute 1-2 μg of DNA in 0.5 ml of reduced serum.
(b) For liposome solution, take 2-25 μl of cationic lipid reagent and dilute to 25 μl, then add 0.5 ml of serum-free medium (serum or antibiotic-free DMEM).
Cationic Lipids
Lipofectamine: 3 : 1 (w/w ) mixture of DOSPA, a multivalent cationic liposome, and DOPE, a neutral liposome (Invitrogen ).
Lipofectin: i.e. cationic liposome DOTMA (N -[l -(2,3 dioleyloxy)-propyl]-N,N ,N -trimethylamonium chloride) mixed with neutral liposome DOPE 1 : 1 (w /w ) (Invitrogen)
LipofectACE: a 1 : 2.5 (w/w ) mixture of synthetic cationic DDAB liposomes and neurolipid DOPE bodies (Invitrogen).
(c) Mix the two solutions together, mix carefully, and then leave for 15-45 min at room temperature to allow the DNA-liposome mixture to form.
4. Rinse the cells with 2 ml of serum-free medium.
5. Spread the DNA-liposome mixture on top of the cultured cells, the transfection reagent should be antibiotic-free.
6. Incubate the transfected cells in a CO2 incubator at 37℃ for 2~24 h, usually 5~6 h.
7. After incubation, discard the transfection mixture and add 3 ml of complete culture medium.
8. 18-24 h later, change the medium (still complete medium).
9. Aspirate the culture medium or harvest the cells according to the requirements of the experiment and measure the transient gene expression activity (see Protocol 27.14 and Protocol 27.15).
B. Transfection of Suspended Cells
1. Prepare the transfection mixture in sterile test tubes as follows:
(a) Dilute 2.5 μg DNA in 0.5 ml of reduced serum medium.
(b) Dilute 2 to 20 μl of cationic liposome reagent in 0.5 ml of serum-free medium.
(c) Mix the two solutions, mix gently, and then incubate at room temperature for 15~45 min to form the DNA-liposome mixture.
2. Centrifuge the suspension of 1X106~2X106 cells and discard the medium.
3. Make another suspension of cells in the transfection mixture and transfer it to a 35 mm dish.
4. Incubate the cells in a CO2 incubator for 4 to 6 h.
5. Add 0.5 ml of medium containing 30% serum to each dish (suspension cells are extremely sensitive to the toxicity of lipid reagents, and a large amount of serum should be added to protect the cells to avoid cell destruction).
6. Incubate overnight in a CO2 incubator.
7. The next day, add 2 ml of complete medium to each culture vessel and continue incubation in a CO2 incubator.
8. 24-72 h after transfection, aspirate the medium or harvest the cells according to the requirements of the experiment, and measure the transient gene expression activity (see Scheme 27.14 and Scheme 27.15).