78
Methods for Setting Up Primary Cultures Specific to Animal Groups
Results
Cells will migrate from the cut opening of the ovarioles (Fig. 4). They
may attach to the bottom of the culture vessel, and form a monolayer
(Kitamura, 1965). They may proliferate to a confluent state. In the case of
ovoviviparous insects, such as flesh fly Sarcophaga peregrina, ovaries
contain developing embryos instead of immature eggs. In this case, cell
migration mainly occurs from embryonic tissues rather than ovarial epithelial tissues, which is the main source of cell migration in lepidopteran
ovaries. Therefore, it may be said that this type of ovarian culture is actually the same as embryonic cell culture. Some cell lines have been established using this method (Kitamura, 1970; Takahashi et aI., 1980).
Subprotocol 4
Imaginal Discs
Like immature insects of other orders, dipteran imaginal discs are also
used as explants for primary culture if they are discernible. In the case of
D. melanogaster, several continuous cell lines have been established from
imaginal discs (Ui et aI., 1987). The following method is based on the
method of Ui et al. for the culture of Drosophila imaginal disc cells.
Materials
Equipment
- Dissecting microscope
- Maximov slide
- Scissors
- Forceps
- Needles
- 24-Well culture dishes (Falcon; Becton-Dickinson)
- Culture tubes (Falcon; Becton-Dickinson)
- Culture vessels
- Pasteur pipette
Solutions - Physiological solution
- Culture medium
Chemicals - Protease VIII (Sigma)
Animals - Larvae of dipteran insects, such as D. melanogaster, preferably reared
aseptically
Procedure
Aseptic 1. Sterilize D. melanogaster eggs in 1/10 diluted saponated cresol solurearing
tion (Japanese Pharmacopoeia) for 10 min, and dechorionate in 2.5%
sodium hypochlorite for 2 min.
Methods for Setting Up Primary Cultures Specific to Animal Groups
Results
Cells will migrate from the cut opening of the ovarioles (Fig. 4). They
may attach to the bottom of the culture vessel, and form a monolayer
(Kitamura, 1965). They may proliferate to a confluent state. In the case of
ovoviviparous insects, such as flesh fly Sarcophaga peregrina, ovaries
contain developing embryos instead of immature eggs. In this case, cell
migration mainly occurs from embryonic tissues rather than ovarial epithelial tissues, which is the main source of cell migration in lepidopteran
ovaries. Therefore, it may be said that this type of ovarian culture is actually the same as embryonic cell culture. Some cell lines have been established using this method (Kitamura, 1970; Takahashi et aI., 1980).
Subprotocol 4
Imaginal Discs
Like immature insects of other orders, dipteran imaginal discs are also
used as explants for primary culture if they are discernible. In the case of
D. melanogaster, several continuous cell lines have been established from
imaginal discs (Ui et aI., 1987). The following method is based on the
method of Ui et al. for the culture of Drosophila imaginal disc cells.
Materials
Equipment
- Dissecting microscope
- Maximov slide
- Scissors
- Forceps
- Needles
- 24-Well culture dishes (Falcon; Becton-Dickinson)
- Culture tubes (Falcon; Becton-Dickinson)
- Culture vessels
- Pasteur pipette
Solutions - Physiological solution
- Culture medium
Chemicals - Protease VIII (Sigma)
Animals - Larvae of dipteran insects, such as D. melanogaster, preferably reared
aseptically
Procedure
Aseptic 1. Sterilize D. melanogaster eggs in 1/10 diluted saponated cresol solurearing
tion (Japanese Pharmacopoeia) for 10 min, and dechorionate in 2.5%
sodium hypochlorite for 2 min.