6 Insecta (Diptera)
79
2. Transfer the eggs to autoclaved Drosophila food (dried yeast-corn
meal-sugar-agar food), and raise at 18°C until the larvae reach the
mature third instar stage.
3. Wash the larvae in sterile distilled water.
1. Dissect the larvae in physiological solution under a dissecting micro- Dissection
scope, and take out the imaginal discs of compound eyes, legs, wings
or antennae (Fig. 5).
2. Remove attached tissues.
1. When many discs have been accumulated (50-300 discs), wash them Dissociation
with fresh physiological solution.
2. Transfer the discs into 1 ml culture medium in a 5-ml culture tube.
3. Add 50 JlI of a 5 mg/ml aqueous solution of protease VIII, and incubate for 1 h at 25°C.
4. Collect still coherent discs, wash in culture medium, and centrifuge at
1,000 g for 1 min.
5. Resuspend the resulting pellet in 4 ml culture medium, and dissociate
by vigorous pipetting.
1. Place 50 JlI of the resulting cell suspension in the center of each well of Culture
a 24-well dish.
set-up
2. After 15-20 min settling time, add 1 ml culture medium to each well.
• • Results
Cells will migrate from the cut opening of the disc fragments (Fig. 6).
Migrated cells first aggregate or sometimes form spherical vesicles
(Fig. 7). After approximately 1 month, proliferation of thin-layered cells
occurs, and the cell population may grow to a continuous cell line. Some
e
d
Fig. 5. Main imaginal discs of Drosophila larva. a eye-antennal disc, b wing disc, c foreleg disc, d middle leg disc,
e hindleg disc, f genitalia disc
79
2. Transfer the eggs to autoclaved Drosophila food (dried yeast-corn
meal-sugar-agar food), and raise at 18°C until the larvae reach the
mature third instar stage.
3. Wash the larvae in sterile distilled water.
1. Dissect the larvae in physiological solution under a dissecting micro- Dissection
scope, and take out the imaginal discs of compound eyes, legs, wings
or antennae (Fig. 5).
2. Remove attached tissues.
1. When many discs have been accumulated (50-300 discs), wash them Dissociation
with fresh physiological solution.
2. Transfer the discs into 1 ml culture medium in a 5-ml culture tube.
3. Add 50 JlI of a 5 mg/ml aqueous solution of protease VIII, and incubate for 1 h at 25°C.
4. Collect still coherent discs, wash in culture medium, and centrifuge at
1,000 g for 1 min.
5. Resuspend the resulting pellet in 4 ml culture medium, and dissociate
by vigorous pipetting.
1. Place 50 JlI of the resulting cell suspension in the center of each well of Culture
a 24-well dish.
set-up
2. After 15-20 min settling time, add 1 ml culture medium to each well.
• • Results
Cells will migrate from the cut opening of the disc fragments (Fig. 6).
Migrated cells first aggregate or sometimes form spherical vesicles
(Fig. 7). After approximately 1 month, proliferation of thin-layered cells
occurs, and the cell population may grow to a continuous cell line. Some
e
d
Fig. 5. Main imaginal discs of Drosophila larva. a eye-antennal disc, b wing disc, c foreleg disc, d middle leg disc,
e hindleg disc, f genitalia disc