Viral Polymerase Activity Assay

1. Seed the DF1 cells in a 12-well plate and culture overnight in DMEM medium. The cell density should be controlled to reach 70-80% confluency the next day when transfection will be conducted.

2. In each well, discard the old medium and supplement with 500 μL of fresh medium before transfection. 

3. Mix 0.6 μg of pPol I-AVI plasmid together with equal amount of Flag-CCT5, or Empty vector in 1.5 mL tube with 200 μL Opti-MEM (serum-free). Incubate at RT for 5 minutes.

3. Mix 2.4 μL of the Lipofectamine 8000 in another 1.5 mL tube 200 uL Opti-MEM. Incubate at RT for 5 minutes.

4. Transfer Mix (3) into Mix (2) and vortex for 10 seconds. Fast spin down the solution from tube wall and cap.

5. Incubate the final mixture for 15 minutes at room temperature.

6. Add the final 400 μL of transfection mixture in a circular dropwise motion to the DF1 cells.

7. Gently swirl the plate in back and forth and side-to-side

8. 12 hours later, discard the transfection solution and wash the cells once with PBS. Then, add 400 ul of FBS-free DMEM with wild type H5N6 influenza virus at an MOI of 0.1.

9. After incubation for 1 hour, remove the virus containing DMEM from the DF1 cells followed by washing 2 times with PBS and adding 1 mL of DMEM containing 10% FBS.

10. After incubating the infected DF1 cells in the incubator for another 12 hours, the cells were washed 2-3 times with PBS and lysed in 100 μL of 1x Passive Lysis buffer.

11. The luciferase activity was measured using the Dual-Luciferase Reporter Assay Kit (Promega E1910 and E1960) according to the instructions.

12. The cell lysates could be also subjected to protein measurement by Western blotting.

Category