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Methods for Setting Up Primary Cultures Specific to Animal Groups
2. Transfer the eggs to autoclaved Drosophila standard food, and rear at
18°C until the larvae reach the mature third instar stage.
3. Wash the larvae in sterile distilled water.
Dissection 1. Dissect the larvae in physiological solution under a dissecting microscope, remove the CNS with forceps, and place it on a Maximov slide
containing culture medium.
Culture 1. Cut the CNS into several pieces.
set-up 2. Treat the fragments with 250 fig/ml protease VIII in the culture medium at 25°C for 1 h.
Treatment
of eggs
Dissociation
3. Wash the fragments once with culture medium containing 10% FBS.
4. Transfer the fragments into a culture vessel with medium conditioned
with D. melanogaster primary cultured embryonic cells.
Results
According to Vi et al. (1994), dissociated CNS cells attached to the bottom of the culture vessels. They consist of various-sized cells. Hollow
spherical vesicles develop occasionally (Fig. 8). In addition, cell aggregates are formed. Fibers connecting the aggregates appear (Fig. 9). The
cells forming the aggregates proliferate and become continuous cell lines.
The cell line obtained with this procedure was identified as a nerve cell
line by its morphology, acetylcholine content, and reactivity to
antihorseradish peroxidase.
Alternative Procedure
This is essentially the same as the procedure for dissociated embryonic
cell cultures. However, during the early embryonic stage, embryos consist mostly of neuronal cells. Thus, by separating the neuronal cells with
a density gradient, an exclusively neuronal cell population can be obtained. The following is based on the method of Hayashi and PerezMagallanes (1994).
1. Allow flies to oviposit for 2 h by supplying standard Drosophila food
to the flies.
2. Keep the oviposited food at 25°C for 3 h until the embryos approach
the early gastrula stage.
3. Collect the eggs, and wash them extensively.
4. Surface sterilize and dechorionate the eggs with a 1 : 1 mixture of 3%
sodium hypochlorite and 95% ethanol.
1. Homogenize the dechorionated embryos with a Dounce homogenizer
in Schneider's medium containing 5% FBS and 200 ng/ml bovine insulin.
Methods for Setting Up Primary Cultures Specific to Animal Groups
2. Transfer the eggs to autoclaved Drosophila standard food, and rear at
18°C until the larvae reach the mature third instar stage.
3. Wash the larvae in sterile distilled water.
Dissection 1. Dissect the larvae in physiological solution under a dissecting microscope, remove the CNS with forceps, and place it on a Maximov slide
containing culture medium.
Culture 1. Cut the CNS into several pieces.
set-up 2. Treat the fragments with 250 fig/ml protease VIII in the culture medium at 25°C for 1 h.
Treatment
of eggs
Dissociation
3. Wash the fragments once with culture medium containing 10% FBS.
4. Transfer the fragments into a culture vessel with medium conditioned
with D. melanogaster primary cultured embryonic cells.
Results
According to Vi et al. (1994), dissociated CNS cells attached to the bottom of the culture vessels. They consist of various-sized cells. Hollow
spherical vesicles develop occasionally (Fig. 8). In addition, cell aggregates are formed. Fibers connecting the aggregates appear (Fig. 9). The
cells forming the aggregates proliferate and become continuous cell lines.
The cell line obtained with this procedure was identified as a nerve cell
line by its morphology, acetylcholine content, and reactivity to
antihorseradish peroxidase.
Alternative Procedure
This is essentially the same as the procedure for dissociated embryonic
cell cultures. However, during the early embryonic stage, embryos consist mostly of neuronal cells. Thus, by separating the neuronal cells with
a density gradient, an exclusively neuronal cell population can be obtained. The following is based on the method of Hayashi and PerezMagallanes (1994).
1. Allow flies to oviposit for 2 h by supplying standard Drosophila food
to the flies.
2. Keep the oviposited food at 25°C for 3 h until the embryos approach
the early gastrula stage.
3. Collect the eggs, and wash them extensively.
4. Surface sterilize and dechorionate the eggs with a 1 : 1 mixture of 3%
sodium hypochlorite and 95% ethanol.
1. Homogenize the dechorionated embryos with a Dounce homogenizer
in Schneider's medium containing 5% FBS and 200 ng/ml bovine insulin.