150
Methods for Setting Up Primary Cultures Specific to Animal Groups
Solutions - Sterile seawater
- Incubation medium
- RPMI-1640 medium 45.5 mg,5% dialyzed FBS,2 ml HEPES/lOO ml
ASW
- Supplement with a "cocktail" of antibiotics (50 mg/ml streptomycin sulfate, 5,104 IV/ml sodium penicillin salt, and 6,250 IV/ml nystatin; 1 ml antibiotic cocktail/IOO ml incubation medium)
- Add collagenase to a final concentration of 0.01 %
- ASW (made of two stock solutions, A and B)
- Stock Solution A: NaCl13.67 g, KCI 0,412 g, and CaCl z ·2H z O 0.721 g
in 125 ml double-distilled water
- Stock Solution B: MgS0 4 ·7HzO 5.57 g and MgCl z ·6H z O 3.05 gin
125 ml double-distilled water
- Mix 125 ml Stock Solution A and 125 ml Stock Solution B, and add
250 ml double-distilled water
- Supplement the mixture with a "cocktail" of antibiotics (50 mg/ml
streptomycin sulfate, 5,104 IV/ml sodium penicillin salt, and
6,250 IV/ml nystatin) at a concentration of 1 ml/100 ml incubation
medium
- Finally, add collagenase (final concentration 0.01 %)
Animals - Six-week-old colonial proto chordate B. schlosseri (for details of the
mariculture of these animals, refer to the section on "Animals" in
subprotocol2)
fIl. Procedure
Dissection 1. Dissect developing buds from well-developed colonies at blastogenic
cycles A-c'
Fig. 6. Epithelial cell sheet formed around the explant of the blastogenic stage B
palleal buds from the colonial protochordate Botryllus schlosseri. b, Explanted fragment of palleal bud. x 100. From Rinkevich, B. and Rabinowitz, c., "Initiation of
epithelial cell cultures from palleal buds of Botryllus schlosseri, a colonial tunicate",
In Vitro Cellular and Developmental Biology-Animal, Vo1.33A, pp,423. Copyright ©
1997 by the Society for In Vitro Biology (formerly the Tissue Culture Association).
Reproduced with permission of the copyright owner
Methods for Setting Up Primary Cultures Specific to Animal Groups
Solutions - Sterile seawater
- Incubation medium
- RPMI-1640 medium 45.5 mg,5% dialyzed FBS,2 ml HEPES/lOO ml
ASW
- Supplement with a "cocktail" of antibiotics (50 mg/ml streptomycin sulfate, 5,104 IV/ml sodium penicillin salt, and 6,250 IV/ml nystatin; 1 ml antibiotic cocktail/IOO ml incubation medium)
- Add collagenase to a final concentration of 0.01 %
- ASW (made of two stock solutions, A and B)
- Stock Solution A: NaCl13.67 g, KCI 0,412 g, and CaCl z ·2H z O 0.721 g
in 125 ml double-distilled water
- Stock Solution B: MgS0 4 ·7HzO 5.57 g and MgCl z ·6H z O 3.05 gin
125 ml double-distilled water
- Mix 125 ml Stock Solution A and 125 ml Stock Solution B, and add
250 ml double-distilled water
- Supplement the mixture with a "cocktail" of antibiotics (50 mg/ml
streptomycin sulfate, 5,104 IV/ml sodium penicillin salt, and
6,250 IV/ml nystatin) at a concentration of 1 ml/100 ml incubation
medium
- Finally, add collagenase (final concentration 0.01 %)
Animals - Six-week-old colonial proto chordate B. schlosseri (for details of the
mariculture of these animals, refer to the section on "Animals" in
subprotocol2)
fIl. Procedure
Dissection 1. Dissect developing buds from well-developed colonies at blastogenic
cycles A-c'
Fig. 6. Epithelial cell sheet formed around the explant of the blastogenic stage B
palleal buds from the colonial protochordate Botryllus schlosseri. b, Explanted fragment of palleal bud. x 100. From Rinkevich, B. and Rabinowitz, c., "Initiation of
epithelial cell cultures from palleal buds of Botryllus schlosseri, a colonial tunicate",
In Vitro Cellular and Developmental Biology-Animal, Vo1.33A, pp,423. Copyright ©
1997 by the Society for In Vitro Biology (formerly the Tissue Culture Association).
Reproduced with permission of the copyright owner