38
General Methods
Equipment
Tissues and
cells
Solutions
Inhibitors
Preparation
of embryonic
cells
Preparation
of blocked
cells
erating cells from tissues difficult to culture. Miltenburger et al. (1985)
developed the following method to obtain a growing cell line from embryonic tissues that were difficult to culture.
Materials
- Stainless steel mesh (0.01 mm 2 )
- Centrifuge and centrifuge tubes
- Hybridoma tissue culture tray (Greiner)
- Forceps
- Two- or three-day-old eggs of the codling moth Cydia pomonella
- IZD-Cp 2202 (a continuous cell line from C. pomonella hemocytes)
- Cp medium (see Appendix 1)
- 2% sodium hypochlorite aqueous solution
- 45% polyethylene glycol (PEG; type 1500; Roth)
- Actinomycin D
- Puromycin
Procedure
1. Surface sterilize approximately 200 eggs of C. pomonella with 2% sodium hypochlorite for 10-15 min.
2. Wash the eggs in 70% ethanol for 3-5 min and then in culture medium.
3. Break the egg shells with a pair of forceps.
4. Pass the contents of the eggs through a stainless steel mesh.
5. Wash the cells twice with serum-free medium by centrifugation at 150 g
for 5 min.
1. Seed approximately 4 x 10 6 cells of IZD-Cp 2202 to two new flasks.
2. Incubate these cells with actinomycin D (0.25 mg/ml) and puromycin
(2 x 10- 4 M), and shake the flasks gently every 15 min for 3 h at 27°C.
3. Wash the cells with serum-free medium twice to remove unbound
inhibitors.
Cell fusion 1. Suspend embryonic cells and IZD-Cp 2202 cells in 5 ml serum-free
Cp medium.
2. Combine these two cell types in one centrifuge tube.
3. Centrifuge at 150 g for 5 min.
4. Suspend the cell pellet in the medium.