36
General Methods
the tissue suspension. Or, if subculturing substrate-dependent cells,
add the dispase solution at 1/10 the volume of the culture medium.
3. Keep the mixture at ambient temperature for 15 min or longer. Prolonged incubation with dispase does not harm tissues or cultured cells.
To stop digestion by dispase, simply dilute the dispase added to the
tissue suspension or cell culture medium with fresh culture medium.
4. Agitate the dispase-containing medium by pipetting.
5. Follow steps 6-7 of the procedure given in subprotocol1.
Results
The results obtained using subprotocol3 are the same as those obtained
using subprotocol 1.
Subprotocol 4
Methods for Subculturing
When the cell number increases sufficiently in a primary culture vessel,
the culture may be split into two or more cultures, or transferred to a
larger vessel. This process is called subculture or passage. The term "subculture" is also used for the culture itself after the first subculture. In a
primary culture, cells usually multiply by attaching themselves to the
vessel. To subculture these cells, the cells should first be detached from
the substrate. To detach cells, partial digestion of the cell surface protein
is usually undertaken. It is also possible to tear off attached cells by scraping them with a rubber policeman, although a considerable number of
cells may be damaged using this method. In the case of a suspended culture or substrate-independent cells, which multiply while they are suspended in the medium, a subculture can be made by simply agitating the
cell suspension by shaking or pipetting to make a homogeneous cell suspension, and then splitting the suspension.
Materials
Equipment - Low-speed centrifuge and centrifuge tubes
- Inverted microscope
Cells - Cultured cells
Solutions - Culture media
- Digesting solution
General Methods
the tissue suspension. Or, if subculturing substrate-dependent cells,
add the dispase solution at 1/10 the volume of the culture medium.
3. Keep the mixture at ambient temperature for 15 min or longer. Prolonged incubation with dispase does not harm tissues or cultured cells.
To stop digestion by dispase, simply dilute the dispase added to the
tissue suspension or cell culture medium with fresh culture medium.
4. Agitate the dispase-containing medium by pipetting.
5. Follow steps 6-7 of the procedure given in subprotocol1.
Results
The results obtained using subprotocol3 are the same as those obtained
using subprotocol 1.
Subprotocol 4
Methods for Subculturing
When the cell number increases sufficiently in a primary culture vessel,
the culture may be split into two or more cultures, or transferred to a
larger vessel. This process is called subculture or passage. The term "subculture" is also used for the culture itself after the first subculture. In a
primary culture, cells usually multiply by attaching themselves to the
vessel. To subculture these cells, the cells should first be detached from
the substrate. To detach cells, partial digestion of the cell surface protein
is usually undertaken. It is also possible to tear off attached cells by scraping them with a rubber policeman, although a considerable number of
cells may be damaged using this method. In the case of a suspended culture or substrate-independent cells, which multiply while they are suspended in the medium, a subculture can be made by simply agitating the
cell suspension by shaking or pipetting to make a homogeneous cell suspension, and then splitting the suspension.
Materials
Equipment - Low-speed centrifuge and centrifuge tubes
- Inverted microscope
Cells - Cultured cells
Solutions - Culture media
- Digesting solution