6 Insecta (Diptera)
81
10. Centrifuge the supernatant at 9,000 g for 30 min at 4°C.
11. Keep the supernatant frozen at -20°C.
12. Remove the precipitate that appears after thawing.
13. Sterilize the extract by passing it through a 0.2-llm pore membrane
filter.
14. Store the extract at -20°C.
Subprotocol 5
Nervous Tissues
In the case of Diptera, nerve tissues oflarvae have been cultured only for
D. melanogaster. There are two ways to culture nerve tissue cells; one
from the CNS of larvae and the other from early embryos. A continuous
cell line has been obtained using the former method (Ui et aI., 1994).
Materials
- Dissecting microscope
- Centrifuge and centrifuge tube
- Dounce homogenizer (Belleo Glass Inc.)
- Celsep apparatus (Brickman Instruments)
- Gradient maker
- Peristaltic pump
- Hemocytometer
- Forceps
- Knife
- Needles
- Maximov slides
- Nytex mesh (25 mm)
- 0.2-llm filter (Nalgen)
- Physiological solution
- Culture medium
- Protease VIII (Sigma)
- Insulin (Sigma)
- Ficoll (Sigma)
- Larvae or eggs of dipterous insects, such as D. melanogaster
Procedure
Equipment
Solutions
Chemicals
Animals
1. Sterilize eggs in 1110 diluted saponated cresol solution (Japanese Phar- Aseptic
macopoeia) for 10 min, and dechorionate in 2.5% sodium hypochlo- rearing
rite for 2 min.