Most of the growth media for cell culture contains Fetal Bovine Serum (FBS). FBS contains protease inhibitors, such as α1-antitrypsin and α2-macroglobulin. These inhibitors stop the trypsin protease and act by inhibiting lysosomal peptidases that may be released. Thus, it is essential to wash the incubated cell with HBSS or PBS to remove FBS. This washing step prevents the inhibition of trypsinisation step.