118
Methods for Setting Up Primary Cultures Specific to Animal Groups
Dissection 1. Make a horse shoe-shaped incision on the abdomen of the grasshoppers.
2. Keep the excised dorsal vessels in culture medium.
3. Remove muscles and fat bodies attached to the dorsal vessels.
Culture 1. Cut the dorsal vessels into small fragments.
set-up 2. Rinse the fragments several times to remove any attached hemocytes.
3. Transfer the tissue fragments into a culture vessel with culture medium, or dispense the cells dispersed from the tissue fragments into a
culture vessel with culture medium.
Results
According to Vago et al. (1968), notwithstanding the washing of the tissue fragments prior to culture, hemocytes appeared soon after the culture was set up. Some hemocytes were attached to the substrate, whereas
others were suspended in the culture medium. On days 5-6 of culture,
many cells had migrated out of the tissue fragments and had formed cell
sheets. These cells survived for several months.
Subprotocol 2
Embryos
Materials
Equipment - Centrifuge and centrifuge tubes
- Beakers
- Forceps
- Small petri dishes (3-5 cm diameter)
- Fine knife
- Needles
- Pasteur pipettes
- T-15 glass flasks (Kontes Glass Co.)
Solutions - Culture medium
Chemicals - Sodium hypochlorite
- Trypsin 1 : 250 (NBC)
Animals - Egg pods of grasshoppers more than 2-3 days after oviposition
Procedure
Sterilization 1. Separate the eggs individually from the egg pods, and clean the eggs
of sand and soil.