310
Related Techniques
Isolation of
single cells
Maintenance
of isolated
cells
2. Follow steps of Preparation of cells and Dilution of cell suspension of
subprotocol 1.
1. Draw up a single cell into the tip of the Pasteur pipette.
2. Cut the tip of the Pasteur pipette containing the single cell.
3. Place the cut tip of the Pasteur pipette into a petri dish.
4. Add culture medium to the dish.
1. Place a lid on the dish and seal the interstice between the lid and the
dish with Parafilm, and keep it at 25°C.
2. When the cells have multiplied in the capillary, break the capillary
and liberate the cells into the petri dish leaving broken glass pieces in
situ, because some cells attached on the inner surface of the capillary.
3. When the cells have multiplied sufficiently to be subcultured, transfer
the cells into a culture flask.
Results
At first, cells will multiply in the capillary. If the cells multiply well, they
may emerge from the opening of the capillary. By breaking the capillary
at this stage, cell proliferation will be promoted further. Suitor et al. (1966)
obtained cell clones using this method.
Subprotocol 3
Oil Overlay Method
Materials
Equipment - Cell-aspirating system (see Comments of subprotocol 1)
- Petri dish
- Pasteur pipettes
- Culture flasks
- Micromanipulator (optional)
- Inverted microscope (see subprotocoll)
Solutions - Cell culture medium
Isolation of
single cells
Sitting
drops
- Cloning medium (see Comments of subprotocol 1)
- Liquid paraffin
Procedure
1. Follow steps of Preparation of cells, Dilution of cell suspension, Capillary and Aspiration of subprotocol 1.
1. Create several sitting drops of cloning medium (approximately 10 fil)
on the bottom of a petri dish.
2. Place the petri dish on a microscope stage.
Related Techniques
Isolation of
single cells
Maintenance
of isolated
cells
2. Follow steps of Preparation of cells and Dilution of cell suspension of
subprotocol 1.
1. Draw up a single cell into the tip of the Pasteur pipette.
2. Cut the tip of the Pasteur pipette containing the single cell.
3. Place the cut tip of the Pasteur pipette into a petri dish.
4. Add culture medium to the dish.
1. Place a lid on the dish and seal the interstice between the lid and the
dish with Parafilm, and keep it at 25°C.
2. When the cells have multiplied in the capillary, break the capillary
and liberate the cells into the petri dish leaving broken glass pieces in
situ, because some cells attached on the inner surface of the capillary.
3. When the cells have multiplied sufficiently to be subcultured, transfer
the cells into a culture flask.
Results
At first, cells will multiply in the capillary. If the cells multiply well, they
may emerge from the opening of the capillary. By breaking the capillary
at this stage, cell proliferation will be promoted further. Suitor et al. (1966)
obtained cell clones using this method.
Subprotocol 3
Oil Overlay Method
Materials
Equipment - Cell-aspirating system (see Comments of subprotocol 1)
- Petri dish
- Pasteur pipettes
- Culture flasks
- Micromanipulator (optional)
- Inverted microscope (see subprotocoll)
Solutions - Cell culture medium
Isolation of
single cells
Sitting
drops
- Cloning medium (see Comments of subprotocol 1)
- Liquid paraffin
Procedure
1. Follow steps of Preparation of cells, Dilution of cell suspension, Capillary and Aspiration of subprotocol 1.
1. Create several sitting drops of cloning medium (approximately 10 fil)
on the bottom of a petri dish.
2. Place the petri dish on a microscope stage.