Cos-1细胞暂时转染protocol

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Note: This protocol has been optimized for Cos-1 Cell s. Successful transfection of each cell type requires optimization of the basic protocol. Variables to consider for optimization include, but are not limited to, cell density, transfection reagent, duration of transfection, and DNA concentration.

1. Seed a six-well tissue culture plate with 1-2 x 105 Cos-1 cells in 2ml of DMEM supplemented with 10% fetal calf serum. Incubate for 18-24 hours at 37¼C in a CO2 humidified incubator or until the cells are 40-60% confluent.

2. Dilute 1-2 micrograms of DNA into 100 microliters serum free DMEM in a sterile tube. To this tube, add 6 microliters of PLUS Reagent found in the LIPOFECTAMINETM PLUS Reagent kit (Life Technologies). Allow mixture to stand for 15 minutes at room temperature. In a second tube dilute 4 microliters of LIPOFECTAMINETM Reagent into 100 microliters serum free DMEM.

3. Combine the two solutions, mixing gently and incubate at room temperature for 10-15 minutes.

4. Wash cells once with 2 ml serum free DMEM .

5. For each transfection, add 0.8 ml serum-free medium to each tube containing the LIPOFECTAMINETM PLUS Reagent - DNA complexes. Mix gently and overlay the complexes onto Cell s.

6. Incubate the cells for 3 hours at 37¼C in a CO2humidified incubator.

7. Remove the DNA-containing medium and replace with 2 ml of DMEM supplemented with 10% fetal calf serum. Incubate cells at 37¼C in a CO2humidified incubator for an additional 48-72 hours.

8. Assay cell extracts for gene expression or activity at 48-72 hours post-transfection.