15 Mollusca
179
Subprotocol 9
Abalone (Gastropoda) Hemocytes
Materials
- Scissors
Equipment
- 5-ml disposable syringe with a 2-G hypodermic needle
- Glass tubes
- 6-Well culture plates
- Modified Alsever's anticoagulant solution (see Appendix 1)
Solutions
- Artificial seawater (ASW)
- Medium-199 modified by Hank by addition of 250 mM NaCI, 10 mM
KCI,25 mM MgS04' 2.5 mM CaCI2, and 10 mM HEPES; pH 7.4; osmolarity 1,100 mOsmol!kg
- Medium-199 modified by Hank containing antifungal and antibacterial substances (100 fig/ml streptomycin sulfate, 60 IU/ml penicillin
G, 50 fig/ml gentamicin sulfate, 0.20 fig/ml amphotericin B, 8 fig/ml
nystatin,2 mM L-glutamine, and 2 mM concanavalin A)
- Abalone Haliotis tuberculata (8-12 cm shell length) maintained in Animals
natural and continuously aerated seawater at ambient temperature with
daily feeding of a mixed-algal diet
Procedure
I. Starve the abalones 2 days prior to use.
2. Make an incision in the foot of the abalone.
3. Collect the hemolymph (8-10 ml) using a 5-ml sterile syringe with a
2-G hypodermic needle.
Bleeding
I. Dilute the hemolymph 1 : 3 with cooled sterile modified Alsever's an- Culture
ticoagulant solution.
set-up
2. Quickly distribute the hemocyte suspension to 6-well culture plates
at a density of 0.8x10 6 hemocytes/well.
3. Add three volume of sterile ASW.
4. Maintain the plates at 15°C.
5. After 90 min, wash the cells with Medium-199 modified by Hank.
6. Replace the medium with one supplemented with antifungal and antibacterial substances.
7. Culture at 15°C.