12 Arthropods Other than Insects
137
1. Mince the tissues with curved iris scissors to make fragments of 0.1- Dissociation
1.0 mm 3 •
2. Incubate the tissue fragments in 200 Vlml type II collagenase. Centrifuge the dissociated cells twice at 50-100 g for 2 min, and wash the
cell pellet each time with fresh medium.
1. Disperse the final pellet into culture medium containing 10 6 IV/ml Culture
penicillin and 100 ~g/ml streptomycin, and culture at 20°e.
set-up
Results
In cultures of testicular tissues, spermatogonial and mesodermal cells
attached to the substrate. Spermatogonial cells persisted in culture for
up to 1 year, and underwent occasional mitosis. Crayfish mesodermal
cells (Fig. 12) persisted longer than one month (Brody and Chang, 1989).
Subprotocol 9
Prawn (Crustacea) Gonad Cells
Materials
- Centrifuge and centrifuge tubes
Equipment
- Scissors
- Forceps
- Pasteur pipettes
- Buffered solution (50% Hank's balanced salt solution, 50% sterile sea- Solutions
water, 1,000 IV/ml penicillin, and 1,000 mgll streptomycin)
- Culture medium (Leibovitz's L-15 medium (see Appendix 1) supplemented with 18% FBS, 30% muscle extract (see Comments), 0.006 glml
NaCI, and 10% lobster hemolymph (Gibco)
- Female adults of the grass prawn Penaeus monodon, weighing approxi- Animals
mately 20 g
Comments
- Muscle extract
- Homogenize approximately 15 g fresh muscle in sterile brackish
water (I : 1 distilled water: sea water)
- Centrifuge the homogenate at 5,000 g for 30 min at 4°C
- Collect the supernatant and filter it through a 0.45-~m pore membrane filter