40 Cell Fusion
371
- Culture medium
Solutions
- PEG Solution
Autoclave 9.4 g PEG (MW = 6,000) for 15 min.
Mix 16 ml serum-free culture medium, 4 ml DMSO,and 0.2 ml of 0.1 %
polyarginine, and sterilize by filtration.
Dissolve the autoclaved PEG into the sterilized mixture.
- DMSO
Chemicals
- Polyarginine (MW = 60,000)
Procedure
1. Prepare well-separated cell suspensions in serum-free culture medium Mixing
from the cell cultures to be fused.
2. Mix approximately the same number of cells from the parent cultures.
3. Centrifuge the mixed cell suspension at 150 g for 5 min.
1. Discard the supernatant, and carefully add 0.25 ml PEG solution.
PEG
2. Stir the mixture gently with a glass rod for 30 s.
treatment
3. Wash the PEG-treated cells with serum-free medium three times by
means of centrifugation at 150 g for 5 min.
4. Suspend the final cell pellet in a serum-containing culture medium,
and culture the cells.
Results
After PEG treatment, residual non -fused parent cells, fused various polyploid homologous, or heterologous cells are present. The efficiency of
the fusion depends on the cell lines used. The concentration of PEG solution and the duration of treatment should be predetermined. Commercially available PEG usually contains substances toxic to cells. It may
be better to choose a suitable PEG from products available from different manufacturers. Procedures for PEG treatment differ somewhat between investigators (Mitsuhashi, 1987).
Subprotocol 3
Electrofusion of Cells
A method using an electric pulse for cell fusion has been devised, and
various electrofusion cell processors are commercially available. In general, the cell fusion process is divided into two phases. The first is
dielectrophoresis, or "pearl chain" formation, induced by an alternating
current, and the second is a decaying pulse, used to make a hole in the
cell membrane with direct current. The second phase is followed by spontaneous repairing of the hole. However, the omission of the first phase
has become common recently because of the development of equipment
371
- Culture medium
Solutions
- PEG Solution
Autoclave 9.4 g PEG (MW = 6,000) for 15 min.
Mix 16 ml serum-free culture medium, 4 ml DMSO,and 0.2 ml of 0.1 %
polyarginine, and sterilize by filtration.
Dissolve the autoclaved PEG into the sterilized mixture.
- DMSO
Chemicals
- Polyarginine (MW = 60,000)
Procedure
1. Prepare well-separated cell suspensions in serum-free culture medium Mixing
from the cell cultures to be fused.
2. Mix approximately the same number of cells from the parent cultures.
3. Centrifuge the mixed cell suspension at 150 g for 5 min.
1. Discard the supernatant, and carefully add 0.25 ml PEG solution.
PEG
2. Stir the mixture gently with a glass rod for 30 s.
treatment
3. Wash the PEG-treated cells with serum-free medium three times by
means of centrifugation at 150 g for 5 min.
4. Suspend the final cell pellet in a serum-containing culture medium,
and culture the cells.
Results
After PEG treatment, residual non -fused parent cells, fused various polyploid homologous, or heterologous cells are present. The efficiency of
the fusion depends on the cell lines used. The concentration of PEG solution and the duration of treatment should be predetermined. Commercially available PEG usually contains substances toxic to cells. It may
be better to choose a suitable PEG from products available from different manufacturers. Procedures for PEG treatment differ somewhat between investigators (Mitsuhashi, 1987).
Subprotocol 3
Electrofusion of Cells
A method using an electric pulse for cell fusion has been devised, and
various electrofusion cell processors are commercially available. In general, the cell fusion process is divided into two phases. The first is
dielectrophoresis, or "pearl chain" formation, induced by an alternating
current, and the second is a decaying pulse, used to make a hole in the
cell membrane with direct current. The second phase is followed by spontaneous repairing of the hole. However, the omission of the first phase
has become common recently because of the development of equipment