~
Single Cell
31 Cell Cloning
311
Fig. 2. Sitting drops of cloning medium
containing single cells and covered with
liquid paraffin
1. Bring the tip of the pipette containing an isolated single cell to above Cell release
one of the drops of medium and release the cell into the drop.
2. Place one cell in each drop of cloning medium.
3. Gently pour autoclaved liquid paraffin over the bottom of the petri
dish so that all drops of cloning medium are covered with the paraffin
{Fig. 2).
4. Keep the petri dish at 25°C.
5. When the cells have multiplied to cover the whole bottom area of the
drop of cloning medium, subculture the cells into a larger vessel. By
repeating the subculture process in this manner, cells can be propagated to establish a cloned population.
Results
The isolated cells may divide. When the cells increase in number, add a
small amount of cloning medium. When the cell number increases sufficiently, transfer the cells to a microtest plate well by pipetting, or by enzyme treatment in the case of substrate-dependent cells. In this manner,
cells will proliferate until flask culture becomes possible.
Subprotocol 4
Fibrin Clot Method
Materials
- Centrifuge and centrifuge tube
Equipment
- Inverted microscope
- 60-Well microtest plate (Falcon; Becton-Dickinson)
- Culture medium
Solutions
- Cloning medium (see Comments of subprotocoll)
- Thrombin solution
- Prepare a 0.001 % thrombin (Nutritional Biochemicals) solution in
distilled water so as to make its activity 1.8 units/ml, and sterilize
the solution by passage through a 0.2-Jlm pore size membrane filter
- Mix 1 ml sterile 0.001 % thrombin solution and 99 ml conditioned
medium
- Mix 10 ml of the thrombin/conditioned medium solution and
140 ml conditioned medium
Single Cell
31 Cell Cloning
311
Fig. 2. Sitting drops of cloning medium
containing single cells and covered with
liquid paraffin
1. Bring the tip of the pipette containing an isolated single cell to above Cell release
one of the drops of medium and release the cell into the drop.
2. Place one cell in each drop of cloning medium.
3. Gently pour autoclaved liquid paraffin over the bottom of the petri
dish so that all drops of cloning medium are covered with the paraffin
{Fig. 2).
4. Keep the petri dish at 25°C.
5. When the cells have multiplied to cover the whole bottom area of the
drop of cloning medium, subculture the cells into a larger vessel. By
repeating the subculture process in this manner, cells can be propagated to establish a cloned population.
Results
The isolated cells may divide. When the cells increase in number, add a
small amount of cloning medium. When the cell number increases sufficiently, transfer the cells to a microtest plate well by pipetting, or by enzyme treatment in the case of substrate-dependent cells. In this manner,
cells will proliferate until flask culture becomes possible.
Subprotocol 4
Fibrin Clot Method
Materials
- Centrifuge and centrifuge tube
Equipment
- Inverted microscope
- 60-Well microtest plate (Falcon; Becton-Dickinson)
- Culture medium
Solutions
- Cloning medium (see Comments of subprotocoll)
- Thrombin solution
- Prepare a 0.001 % thrombin (Nutritional Biochemicals) solution in
distilled water so as to make its activity 1.8 units/ml, and sterilize
the solution by passage through a 0.2-Jlm pore size membrane filter
- Mix 1 ml sterile 0.001 % thrombin solution and 99 ml conditioned
medium
- Mix 10 ml of the thrombin/conditioned medium solution and
140 ml conditioned medium