31 Cell Cloning
319
- Culture plate insert (Fig.4; cell culture inserts for 6-, 12-, or 24- well
culture plates are commercially available; e.g. Falcon, cell culture insert, Becton-Dickinson; Transwell, Corning)
- Culture medium
Procedure
1. Select a culture whose cells to be used for the nurse culture and suspend the cells in the culture medium.
2. Centrifuge the cells at 150 g for 5 min.
3. Suspend the cell pellet in culture medium.
4. Distribute the cell suspension into each well of a 24-well culture plate.
1. Place a culture plate insert for 24-well culture plate in each well containing nurse cells.
2. Dispense a small amount of culture medium just cover the bottom of
the insert into each culture plate insert.
3. Select a culture whose cells to be cloned.
4. Follow the procedures given in subprotocol 1 for the Preparation of
cells, Dilution of cell suspension, Preparation of a capillary and Aspiration of cells.
5. Liberate the isolated cell into each culture plate insert.
6. Culture the isolated cell until it multiplies and can be subcultured.
Results
The isolated cells will multiply by receiving growth factor from the nurse
cells through the membrane of the culture plate insert. However, it is
difficult to observe the cells inside the culture plate insert under a microscope.
Comments
It is also possible to place a single cell in the well of a culture plate, and to
place a culture plate insert containing nurse cells into the well.
References
Solutions
Preparation
of nurse
culture
Culture of
isolated
single cells
Itoh, T. and Mitsuhashi, J. (1995) Effects of various conditioned media on proliferation of an isolated single cell from insect cell lines. In Vitro Cell. Dev. Biol. 32A, 814-816.
Mcintosh, A.H. and Rectoris, C. (1974) Insect cells: colony formation and cloning in agar medium.
In Vitro 10, 1-5.
Mitsuhashi, J. (1975) Isolation of a clone from an insect cell line by application of a fibrin clot
method. Appl. Entomol. Zool. 10,309-310.
Peel, D.J. and Milner, M.J. (1990) The diversity of cell morphology in cloned cell lines derived from
Drosophila imaginal discs. Roux's Arch. Dev. Biol. 198,479-482.
Suitor, E.C. Jr., Chang, L.L. and Liu, H.H. (1966) Establishment and characterization of a clone from
Grace's in vitro cultured mosquito (Aedes aegypti L.) cells. Exp. Cell Res. 44, 572-578.
319
- Culture plate insert (Fig.4; cell culture inserts for 6-, 12-, or 24- well
culture plates are commercially available; e.g. Falcon, cell culture insert, Becton-Dickinson; Transwell, Corning)
- Culture medium
Procedure
1. Select a culture whose cells to be used for the nurse culture and suspend the cells in the culture medium.
2. Centrifuge the cells at 150 g for 5 min.
3. Suspend the cell pellet in culture medium.
4. Distribute the cell suspension into each well of a 24-well culture plate.
1. Place a culture plate insert for 24-well culture plate in each well containing nurse cells.
2. Dispense a small amount of culture medium just cover the bottom of
the insert into each culture plate insert.
3. Select a culture whose cells to be cloned.
4. Follow the procedures given in subprotocol 1 for the Preparation of
cells, Dilution of cell suspension, Preparation of a capillary and Aspiration of cells.
5. Liberate the isolated cell into each culture plate insert.
6. Culture the isolated cell until it multiplies and can be subcultured.
Results
The isolated cells will multiply by receiving growth factor from the nurse
cells through the membrane of the culture plate insert. However, it is
difficult to observe the cells inside the culture plate insert under a microscope.
Comments
It is also possible to place a single cell in the well of a culture plate, and to
place a culture plate insert containing nurse cells into the well.
References
Solutions
Preparation
of nurse
culture
Culture of
isolated
single cells
Itoh, T. and Mitsuhashi, J. (1995) Effects of various conditioned media on proliferation of an isolated single cell from insect cell lines. In Vitro Cell. Dev. Biol. 32A, 814-816.
Mcintosh, A.H. and Rectoris, C. (1974) Insect cells: colony formation and cloning in agar medium.
In Vitro 10, 1-5.
Mitsuhashi, J. (1975) Isolation of a clone from an insect cell line by application of a fibrin clot
method. Appl. Entomol. Zool. 10,309-310.
Peel, D.J. and Milner, M.J. (1990) The diversity of cell morphology in cloned cell lines derived from
Drosophila imaginal discs. Roux's Arch. Dev. Biol. 198,479-482.
Suitor, E.C. Jr., Chang, L.L. and Liu, H.H. (1966) Establishment and characterization of a clone from
Grace's in vitro cultured mosquito (Aedes aegypti L.) cells. Exp. Cell Res. 44, 572-578.