31 Cell Cloning
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1. Keep the plate in a moist chamber to avoid desiccation (100 % humid- Maintenance
ity; kept by placing sterile absorbent cotton soaked in sterile distilled
water along the bottom corner of the chamber).
2. Ascertain the existence of a single cell in the microtest plate well.
3. When cells have multiplied to cover the whole bottom area of the
micro test plate well, subculture the cells into a larger vessel. By repeating the subculture procedure in this manner, cells can be propagated to establish a cloned population.
Comments
A simple cell-aspirating system consists of a capillary connected with
rubber tubing to a mouth part. However, aspiration using the mouth pipette is associated with a risk of contamination by mycoplasmas. Alternatively, an electrically operated cell-aspiration system may be used.
A nutrient-rich medium is recommended for use as the cloning medium. Often, a 1 : 1 mixture of fresh medium and conditioned medium is
used.
Results
Isolated cells may divide in the well of a microtest plate. However, synchronization of the cell cycle of dividing cells will not continue for long.
The size and shape of the divided cells also fluctuates. It is not necessary
to use a conditioned medium with homologous cell culture. The ability
of conditioned medium to promote cell growth depends on the cell line
used for to condition the medium (Itoh and Mitsuhashi, 1995).
Subprotocol 2
Capillary Method
Materials
- Inverted microscope (see subprotocoll)
- Micromanipulator (optional)
- Pasteur pipettes
- Cell aspirating system (see Comments of subprotocoll)
- Petri dish
- Culture flasks
- Culture medium
Procedure
Equipment
Solutions
1. Draw the tip of a Pasteur pipette out over a flame (see Capillary steps Pretreatment
1-2 of subprotocoll).
309
1. Keep the plate in a moist chamber to avoid desiccation (100 % humid- Maintenance
ity; kept by placing sterile absorbent cotton soaked in sterile distilled
water along the bottom corner of the chamber).
2. Ascertain the existence of a single cell in the microtest plate well.
3. When cells have multiplied to cover the whole bottom area of the
micro test plate well, subculture the cells into a larger vessel. By repeating the subculture procedure in this manner, cells can be propagated to establish a cloned population.
Comments
A simple cell-aspirating system consists of a capillary connected with
rubber tubing to a mouth part. However, aspiration using the mouth pipette is associated with a risk of contamination by mycoplasmas. Alternatively, an electrically operated cell-aspiration system may be used.
A nutrient-rich medium is recommended for use as the cloning medium. Often, a 1 : 1 mixture of fresh medium and conditioned medium is
used.
Results
Isolated cells may divide in the well of a microtest plate. However, synchronization of the cell cycle of dividing cells will not continue for long.
The size and shape of the divided cells also fluctuates. It is not necessary
to use a conditioned medium with homologous cell culture. The ability
of conditioned medium to promote cell growth depends on the cell line
used for to condition the medium (Itoh and Mitsuhashi, 1995).
Subprotocol 2
Capillary Method
Materials
- Inverted microscope (see subprotocoll)
- Micromanipulator (optional)
- Pasteur pipettes
- Cell aspirating system (see Comments of subprotocoll)
- Petri dish
- Culture flasks
- Culture medium
Procedure
Equipment
Solutions
1. Draw the tip of a Pasteur pipette out over a flame (see Capillary steps Pretreatment
1-2 of subprotocoll).