Chapter 31
Cell Cloning
Most continuous insect cell lines consist of a heterogeneous cell population, probably because the cell lines originate from explants that themselves consist of various tissues or cell types. In addition, it is true that a
cell line becomes heterogeneous with repeated passaging. However, it is
desirable to have a more homogeneous cell population for use as experimental material. To this end, cell cloning is useful. However, cloned insect cell lines often become heterogeneous again within a short period
of time. In this case, recloning or repeated cloning may help to unify the
cell population. Cloning is essentially a method of isolating single cells
and proliferating the isolated cells. There are several methods by which
clones can be obtained.
Subprotocol 1
Single Cell Cloning
Materials
PROTOCOL
- Inverted microscope (a simple microscope with a phase-contrast op- Equipment
tical system is preferable)
- Micromanipulator (optional)
- Pasteur pipettes
- Cell-aspirating system (see Comments)
- Microtest plates (e.g., Terasaki plate; Nunc)
- Moist chamber (an air-tight plastic box is preferable)
- Cloning medium (see Comments)
- 0.1 % Benzalkonium chloride
- Parafilm (American National Can) or similar
Procedure
1. Harvest the cells.
2. Centrifuge the cell suspension at 150 g for 5 min.
3. Disperse the cell pellet in cloning medium.
Solutions
Miscellaneous
Preparation
of cells
Cell Cloning
Most continuous insect cell lines consist of a heterogeneous cell population, probably because the cell lines originate from explants that themselves consist of various tissues or cell types. In addition, it is true that a
cell line becomes heterogeneous with repeated passaging. However, it is
desirable to have a more homogeneous cell population for use as experimental material. To this end, cell cloning is useful. However, cloned insect cell lines often become heterogeneous again within a short period
of time. In this case, recloning or repeated cloning may help to unify the
cell population. Cloning is essentially a method of isolating single cells
and proliferating the isolated cells. There are several methods by which
clones can be obtained.
Subprotocol 1
Single Cell Cloning
Materials
PROTOCOL
- Inverted microscope (a simple microscope with a phase-contrast op- Equipment
tical system is preferable)
- Micromanipulator (optional)
- Pasteur pipettes
- Cell-aspirating system (see Comments)
- Microtest plates (e.g., Terasaki plate; Nunc)
- Moist chamber (an air-tight plastic box is preferable)
- Cloning medium (see Comments)
- 0.1 % Benzalkonium chloride
- Parafilm (American National Can) or similar
Procedure
1. Harvest the cells.
2. Centrifuge the cell suspension at 150 g for 5 min.
3. Disperse the cell pellet in cloning medium.
Solutions
Miscellaneous
Preparation
of cells