To neutralize trypsin after detaching cells from the culture surface, you can add an equal volume of culture medium containing serum or a trypsin inhibitor. This step is crucial to prevent the continued degradation of cellular proteins by trypsin, which can compromise cell viability and function.
- Serum: Serum, such as fetal bovine serum (FBS), is a common component of cell culture media that provides growth factors, hormones, and nutrients for cells. Serum also contains proteins like alpha-2-macroglobulin and other protease inhibitors that can neutralize trypsin’s activity. When an equal volume of serum-containing medium is added to the cell suspension after trypsin treatment, trypsin is rapidly neutralized, protecting the cells from further damage.
- Trypsin inhibitors: An alternative to using serum-containing medium is to use a specific trypsin inhibitor, such as soybean trypsin inhibitor (SBTI) or phenylmethylsulfonyl fluoride (PMSF). These inhibitors can be added directly to the trypsinized cell suspension to neutralize trypsin activity. The concentration of the trypsin inhibitor depends on the specific inhibitor used and the experimental conditions, so consult the manufacturer’s instructions or relevant literature for the appropriate concentration.
After neutralizing the trypsin, gently pipette the cell suspension up and down to break up any cell clumps and transfer the cells to a new culture vessel or centrifuge tube as needed. Make sure to maintain aseptic conditions and monitor cell growth and viability following passaging.
