312
Related Techniques
Dilution of
the cell
suspension
Fibrin clot
formation
- Bovine fibrinogen solution
- Prepare a 0.5% fibrinogen (Sigma) solution in saline (800 mg NaCl,
50 mg KCI, and 20 mg Na3C6Hs07·2H20 in 100 ml distilled water),
and sterilize the solution by passage through a 0.2-llm pore size
membrane filter
Procedure
1. Select a cell culture, and suspend the cells in the culture medium.
2. Centrifuge the cell suspension at 150 g for 5 min.
3. Suspend the cell pellet in the thrombin solution so as to make a final
cell density of 10 cells/ml.
1. Mix 12 ml of the above cell suspension and 3 ml fibrinogen solution.
2. Distribute 0.1 ml of the cell suspension into each well of a microtest
plate.
3. Examine each well under a microscope, and mark the wells that contain a single cell.
4. When the cells have multiplied to cover the whole bottom area of the
well, subculture the cells with fresh medium without thrombin to a
larger vessel. By repeating the subculture process in this manner, cells
may be propagated to establish a cloned population.
Results
A fibrin clot will be formed within 24 h. The isolated cells will proliferate
in the fibrin clot. Mitsuhashi (1975) obtained a clone from a mixed population of the NIAS-PX -58 cell line from the swallow tail butterfly Papilio
xuthus.
Subprotocol 5
Colony Formation for Substrate-Dependent Cells
Materials
Equipment - Centrifuge and centrifuge tube
- Culture plate
- Petri dish
- Cloning cylinders (stainless steel, glass, or polystyrene rings; inner
diameter approximately 5 mm, thickness 2-3 mm, height 8-10 mm;
e.g., Sigma cloning cylinder)
Solutions - Culture medium
- Cloning medium (see Comments of subprotocoll)
Related Techniques
Dilution of
the cell
suspension
Fibrin clot
formation
- Bovine fibrinogen solution
- Prepare a 0.5% fibrinogen (Sigma) solution in saline (800 mg NaCl,
50 mg KCI, and 20 mg Na3C6Hs07·2H20 in 100 ml distilled water),
and sterilize the solution by passage through a 0.2-llm pore size
membrane filter
Procedure
1. Select a cell culture, and suspend the cells in the culture medium.
2. Centrifuge the cell suspension at 150 g for 5 min.
3. Suspend the cell pellet in the thrombin solution so as to make a final
cell density of 10 cells/ml.
1. Mix 12 ml of the above cell suspension and 3 ml fibrinogen solution.
2. Distribute 0.1 ml of the cell suspension into each well of a microtest
plate.
3. Examine each well under a microscope, and mark the wells that contain a single cell.
4. When the cells have multiplied to cover the whole bottom area of the
well, subculture the cells with fresh medium without thrombin to a
larger vessel. By repeating the subculture process in this manner, cells
may be propagated to establish a cloned population.
Results
A fibrin clot will be formed within 24 h. The isolated cells will proliferate
in the fibrin clot. Mitsuhashi (1975) obtained a clone from a mixed population of the NIAS-PX -58 cell line from the swallow tail butterfly Papilio
xuthus.
Subprotocol 5
Colony Formation for Substrate-Dependent Cells
Materials
Equipment - Centrifuge and centrifuge tube
- Culture plate
- Petri dish
- Cloning cylinders (stainless steel, glass, or polystyrene rings; inner
diameter approximately 5 mm, thickness 2-3 mm, height 8-10 mm;
e.g., Sigma cloning cylinder)
Solutions - Culture medium
- Cloning medium (see Comments of subprotocoll)