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Related Techniques
Subprotocol 8
Feeder Layer Culture
Materials
Equipment - X-ray or y-ray irradiation apparatus
- Centrifuge and centrifuge tubes
- Petri dishes (3 cm diameter)
- Pasteur pipette
- Culture flasks
Solutions - Culture medium
Freeder
layer
formation
Irradiation
Seeding
Procedure
1. Prepare the culture whose cells to be used for making the feeder layer.
2. Suspend the cells in the medium, and centrifuge the cells at 150 g for
5 min.
3. Suspend the cells in culture medium.
4. Distribute the cell suspension to petri dishes.
5. Culture the cells until confluency is attained.
1. Remove the culture medium from the petri dishes.
2. Irradiate the cells with an X-ray ory-ray (the strength of the radiation
and the duration of irradiation should be determined by preliminary
experiments) .
3. Add the culture medium, and continue the culture.
1. Select a culture whose cells to be cloned, and harvest the cells from
the culture.
2. Follow the procedures given in subprotocol 1 for Dilution of the cell
suspension, Preparation of the capillary and Aspiration of cells.
3. Liberate the isolated cell into a petri dish containing the irradiated
cell monolayer.
Maintenance 1. Culture the cell at the standard temperature for the cell.
2. Harvest the cells when they have multiplied sufficiently.
3 Transfer these cells to a culture flask.
4. When the cells have multiplied to cover the whole bottom area of the
well, subculture them to a larger vessel. By repeating the subculture
process in this manner, cells may be propagated to establish a cloned
population.