372
Related Techniques
able to apply a high-voltage uniform electric field and the use of highdensity cell suspensions (higher than 10 8 cells/ml). There are two types
of electrodes used in electrofusion. One is a micro electrode that consists
of pin-points of platinum electrode or glass capillaries. This electrode
can be set by means of a micromanipulator so as to place the cells to be
fused between the electrodes. Thus, it is possible to select the cell pair to
be fused, and the process of fusion can be followed under a microscope.
The other type of electrode is the parallel electrode, which consists of
platinum plates placed parallel to each other, leaving a gap of 0.1 to several mm between the electrodes. With this type of electrode, it is possible to fuse many cell pairs simulataneously. The latter type of electrode
is commonly used.
Materials
Equipment - Electrofusion cell processor (e.g., EMC 2001, BTX)
- Centrifuge and centrifuge tubes
- Pasteur pipettes
Solutions - Physiological solution
Preparation
of cells
Dielectrophoresis
- Chamber medium (0.3 M mannitol containing 2.5 mM MgC1 2 ; sorbitol or glucose may be used instead of mannitol)
- Culture medium
Procedure
1. Harvest cells from the two cell lines to be fused.
2. Wash the cells from each cell line three times with physiological solution by centrifugation at 150 g for 5 min.
3. Suspend the cells from each cell line in chamber medium.
4. Wash the cells from each cell line with chamber medium by centrifugation at 150 g for 5 min.
5. Mix cell suspensions of the cell lines to be fused at an appropriate
ratio.
6. Centrifuge the mixed cell suspension at 150 g for 5 min.
7. Resuspend the pellet in chamber medium so that the cell density is
approximately 10 7 cells/ml.
1. Place the mixed cell suspension in an electrofusion chamber.
2. Wait for 5 min, allowing the cells to form a sediment on the bottom of
the chamber.
3. Apply alternating current at 600 kHz, 50 V /cm. This causes the cells to
become attracted to one electrode and results in the production of
long pearl chains (Fig. O. If the voltage is increased to higher than
100 V / em, the cells will start to rotate and, at higher voltages, the cells
will burst.
Related Techniques
able to apply a high-voltage uniform electric field and the use of highdensity cell suspensions (higher than 10 8 cells/ml). There are two types
of electrodes used in electrofusion. One is a micro electrode that consists
of pin-points of platinum electrode or glass capillaries. This electrode
can be set by means of a micromanipulator so as to place the cells to be
fused between the electrodes. Thus, it is possible to select the cell pair to
be fused, and the process of fusion can be followed under a microscope.
The other type of electrode is the parallel electrode, which consists of
platinum plates placed parallel to each other, leaving a gap of 0.1 to several mm between the electrodes. With this type of electrode, it is possible to fuse many cell pairs simulataneously. The latter type of electrode
is commonly used.
Materials
Equipment - Electrofusion cell processor (e.g., EMC 2001, BTX)
- Centrifuge and centrifuge tubes
- Pasteur pipettes
Solutions - Physiological solution
Preparation
of cells
Dielectrophoresis
- Chamber medium (0.3 M mannitol containing 2.5 mM MgC1 2 ; sorbitol or glucose may be used instead of mannitol)
- Culture medium
Procedure
1. Harvest cells from the two cell lines to be fused.
2. Wash the cells from each cell line three times with physiological solution by centrifugation at 150 g for 5 min.
3. Suspend the cells from each cell line in chamber medium.
4. Wash the cells from each cell line with chamber medium by centrifugation at 150 g for 5 min.
5. Mix cell suspensions of the cell lines to be fused at an appropriate
ratio.
6. Centrifuge the mixed cell suspension at 150 g for 5 min.
7. Resuspend the pellet in chamber medium so that the cell density is
approximately 10 7 cells/ml.
1. Place the mixed cell suspension in an electrofusion chamber.
2. Wait for 5 min, allowing the cells to form a sediment on the bottom of
the chamber.
3. Apply alternating current at 600 kHz, 50 V /cm. This causes the cells to
become attracted to one electrode and results in the production of
long pearl chains (Fig. O. If the voltage is increased to higher than
100 V / em, the cells will start to rotate and, at higher voltages, the cells
will burst.