Begin by maintaining HEK293T cells and DMEM containing heat inactivated FBS, Penicillin, and Streptomycin in a T75 flask. For cell counting, mix 20 microliters of cell suspension with 20 microliters of trypan blue solution. Add 20 microliters of dye-mixed cell suspension to the cell counting chamber and count the number of cells per milliliter.
Seed HEK293T cells and 10 milliliters of complete DMEM in a 10 centimeter cell culture Petri dish. Incubate the cells overnight in a carbon dioxide incubator at 37 degrees Celsius. The next day, remove the complete medium and replace it with nine milliliters of DMEM serum-free medium.
Using the given compounds of viral particle assembly, prepare a cotransfection mixture. Slowly add the mixture to the Petri dish, and incubate at 37 degrees Celsius for six hours. After incubation, replace the serum-free DMEM with a complete DMEM medium and continue incubation for cotransfection for up to 48 hours.
Then detach the cells by repeatedly pipetting over the surface of the monolayer and transfer the suspension into a 15 milliliter tube. Centrifuge the cell suspension at 400G for five minutes. Collect the supernatant and pass it through a 0.22 micron filter.
Store the filtrate containing Ha-CoV-2 pseudovirus at 80 degrees Celsius.
