TPCK-treated trypsin is available from several manufacturers. It is available in varying concentrations from different manufacturers, and some manufacturers offer the product based on specific activity, not concentration. Additionally, concentration will depend on purity. Therefore, it is important to determine the appropriate concentration of each lot of TPCK-trypsin used in the lab. To determine the appropriate concentration, the host cells should first be at 100% confluency before TPCK-treated trypsin is added. Then various concentrations should be tested on the 100% confluent host cell monolayer to determine the highest concentration that can be added to the cells without causing cytotoxicity to the host cell monolayer for the duration of the incubation period needed for growth of the virus. This testing should be conducted for each cell line that may be used with TPCK-treated trypsin to grow influenza virus, and must be repeated with each new lot of TPCK-treated trypsin.