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Methods for Setting Up Primary Cultures Specific to Animal Groups
Subprotocol 1 S
Neurons of the Nudibranches (Opistobranchia)
Materials
Equipment - Culture dish (no. 3001; Falcon, Becton-Dickinson)
- No.1 round glass cover slip coated with poly L-Iysine (MW > 300,000;
Sigma)
- Petri dish
- Microcentrifuge tube (1.5 ml)
Solutions - Nudibranch culture medium (see Appendix 1)
- ASW (composition of ASW (in mM): NaC1465; KCII0.5; MgCl 2 ·6H 2 0
54.2; MgS0 4 ·7H 2 0 1.7; CaCI 2 ·2H 2 0 11.0; glucose 11.0; HEPES 15.0; PenStrep 50 U/50 Ilg/ml)
- Bovine serum albumin solution (BSA; 180 mg/IO ml ASW)
Animals - Nudibranch Heremissenda crassicornis
Procedure
Sterilization 1. Spray the dorsal surface of the animal quickly with 70% ethanol.
Dissection 1. Dissect out a circumesophageal nerve ring (Fig. 21).
2. Rinse the nerve ring in fresh ASW.
Digestion 1. Digest the nerve ring in a sterile 1.5-ml micro centrifuge tube containing 25 mg protease dissolved in 1 ml ASW.
2. Continue the digestion overnight (approximately 17 h) at 12°C.
3. Transfer the nerve ring to BSA solution (180 mg BSA/lO ml ASW).
4. Renew the BSA solution after 15 min.
5. After a further 15 min, wash the nerve ring in ASW for 30 min.
Dissection 1. Cut the nerve ring into four fragments, and dissociate the neuronal
cells.
Culture 1. Transfer the isolated cells into a dish containing culture medium, and
set-up
leave for 5 min.
2. Move the dish to a 12°C incubator and keep the culture undisturbed
for 24 h.
Results
According to Tamse et al. (1995), cultured neurons extend neurite processes within 5 h of isolation. These neurons showed various morphology. In addition to neuronal cells, cells that seemed to be glial cells also
grew in culture. The neuronal cells growing in close proximity with each
Methods for Setting Up Primary Cultures Specific to Animal Groups
Subprotocol 1 S
Neurons of the Nudibranches (Opistobranchia)
Materials
Equipment - Culture dish (no. 3001; Falcon, Becton-Dickinson)
- No.1 round glass cover slip coated with poly L-Iysine (MW > 300,000;
Sigma)
- Petri dish
- Microcentrifuge tube (1.5 ml)
Solutions - Nudibranch culture medium (see Appendix 1)
- ASW (composition of ASW (in mM): NaC1465; KCII0.5; MgCl 2 ·6H 2 0
54.2; MgS0 4 ·7H 2 0 1.7; CaCI 2 ·2H 2 0 11.0; glucose 11.0; HEPES 15.0; PenStrep 50 U/50 Ilg/ml)
- Bovine serum albumin solution (BSA; 180 mg/IO ml ASW)
Animals - Nudibranch Heremissenda crassicornis
Procedure
Sterilization 1. Spray the dorsal surface of the animal quickly with 70% ethanol.
Dissection 1. Dissect out a circumesophageal nerve ring (Fig. 21).
2. Rinse the nerve ring in fresh ASW.
Digestion 1. Digest the nerve ring in a sterile 1.5-ml micro centrifuge tube containing 25 mg protease dissolved in 1 ml ASW.
2. Continue the digestion overnight (approximately 17 h) at 12°C.
3. Transfer the nerve ring to BSA solution (180 mg BSA/lO ml ASW).
4. Renew the BSA solution after 15 min.
5. After a further 15 min, wash the nerve ring in ASW for 30 min.
Dissection 1. Cut the nerve ring into four fragments, and dissociate the neuronal
cells.
Culture 1. Transfer the isolated cells into a dish containing culture medium, and
set-up
leave for 5 min.
2. Move the dish to a 12°C incubator and keep the culture undisturbed
for 24 h.
Results
According to Tamse et al. (1995), cultured neurons extend neurite processes within 5 h of isolation. These neurons showed various morphology. In addition to neuronal cells, cells that seemed to be glial cells also
grew in culture. The neuronal cells growing in close proximity with each