Hello, I am currently establishing a virus infection model in my lab. The cell I use is a vero cell and the virus is porcine epidemic diarrhea virus (PDEV). I plan to use the virus stock without concentration titration to make the new virus stocks, and I will conduct TCID50 analysis of the virus stocks in the future.

First, 0.5 ug/ml to 2 ug/ml TPCK-trypsin test was conducted in 96 well for the condition of virus infection culture (including 0.3% BSA). The virus infection culture was treated after washing twice with plain DMEM using 80-90% vero cells (Figure 1). Then, the appropriate concentrations (0.5 ug/ml, 0.75 ug/ml, 1 ug/ml) were selected and PEDV infection was performed.

Similar to the above process, after washing twice with plain DMEM, the virus stock of unknown concentration was diluted by virus infection culture (by TPCK-trpysin concentration) at 1:10. Finally 5 ml was dispensed into 100 dishes for 1 hour incubation. Next, I washed it twice with plain DMEM, added 10ml of the virus-infected culture medium, and incubated it for 3 days (Figure 2).

What I am curious about is the concentration (0.5 ug/ml or 0.75 ug/ml?) of TPCK-trypsin to establish a virus infection model and the timepoint of harvesting cell supernatant for virus stock manufacturing. What state should the vero cell be in to manufacture the virus stock? Please advise us to establish a virus infection model. Thank you!