Chapter 4
General Cell Culture Methods
Subprotocol 1
Trypsin Treatment for Dissociation of Cell Masses
Trypsin is the most widely used enzyme in mammalian cell culture. However, it cannot be used for the culture of some insect cells because of
their sensitivity to it. The action of trypsin is inhibited by the presence of
Mg2+ and Ca 2 +. Therefore, trypsin should be dissolved in a physiological
solution free of Mg2+ and Ca 2 +. Rinaldini's solution (Rinaldini, 1959; see
Appendix 1) is often used as one such solution. Balanced salt solution
(BSS) or phosphate-buffered saline (PBS) from which Mg2+ and Ca2+ have
been omitted can also be used. The optimum pH for trypsin is 8-9.
Trypsin is often used with ethylenediamine tetraacetic acid (EDTA).
Materials
- Centrifuge and centrifuge tubes
- Magnetic stirrer and magnetic bar
- Stainless steel mesh (150 mesh)
- Incubator
PROTOCOL
Equipment
- Rinaldini's solution (see Appendix 1)
Solutions
- Trypsin solution
Dissolve 0.1 g trypsin (e.g. Difco Laboratories, Trypsin 1 : 250) in
100 ml Rinaldini's solution. The trypsin powder does not dissolve immediately in water. Keep the trypsin solution that has not yet dissolved
completely in a refrigerator overnight and continue stirring the solution by means of a magnetic stirrer.
Dissolve 0.02 g EDTA in 100 ml Rinaldini's solution.
When the trypsin solution becomes clear, mix both the trypsin and
EDTA solutions in a ratio of 1 : 1.
Store the filter-sterilized trypsin-EDTA solution below -20°C until
used.
- Culture media