Chapter 13
Prochordata
Subprotocol 1
Pharyngeal Tissues of the Solitary Protochordates (Tunicata)
Circulating hemocytes of the solitary proto chordates do not survive for
prolonged periods in vitro. Subsequently, pharyngeal tissue, which is considered a hemocytopoietic tissue, has been cultured by Raftos et al. (1990).
The following procedure is based on their method.
Materials
- Glass aquarium
- Centrifuge and centrifuge tubes
- Scissors
- Surgical knife
- Forceps
- 24-Well tissue culture plate
PROTOCOL
Equipment
- Artificial seawater (ASW; 3.4% w/v; Instant Ocean; Aquarium System) Solutions
- Tunicate tissue culture medium (TTCM)
454 mg RPMI-1640 powder, 40 mg streptomycin sulfate, 10,000 IV
penicillin sulfate, 20% (v/v) tunic ate plasma (see Comments)/1,OOO ml
ASW.
- A tunicate Styela clava can be purchased from Marinus
Animals
- Marine invertebrate diet (Hawaiian Marine Imports)
Miscellaneous
Procedure
1. Maintain tunicates in a glass aquarium filled with ASW by supplying
the invertebrate diet every 2 days.
l. Dissect the animals and remove pharyngeal tissues (Fig. 1).
2. Cut the tissues into 3x3 mm fragments.
1. Place one fragment per well of 24-well tissue culture plates containing
1 ml TTCM/well.
2. Seal the plate and keep at 15°C in normal atmosphere.
Maintenance
of animals
Dissection
Culture
set-up
Prochordata
Subprotocol 1
Pharyngeal Tissues of the Solitary Protochordates (Tunicata)
Circulating hemocytes of the solitary proto chordates do not survive for
prolonged periods in vitro. Subsequently, pharyngeal tissue, which is considered a hemocytopoietic tissue, has been cultured by Raftos et al. (1990).
The following procedure is based on their method.
Materials
- Glass aquarium
- Centrifuge and centrifuge tubes
- Scissors
- Surgical knife
- Forceps
- 24-Well tissue culture plate
PROTOCOL
Equipment
- Artificial seawater (ASW; 3.4% w/v; Instant Ocean; Aquarium System) Solutions
- Tunicate tissue culture medium (TTCM)
454 mg RPMI-1640 powder, 40 mg streptomycin sulfate, 10,000 IV
penicillin sulfate, 20% (v/v) tunic ate plasma (see Comments)/1,OOO ml
ASW.
- A tunicate Styela clava can be purchased from Marinus
Animals
- Marine invertebrate diet (Hawaiian Marine Imports)
Miscellaneous
Procedure
1. Maintain tunicates in a glass aquarium filled with ASW by supplying
the invertebrate diet every 2 days.
l. Dissect the animals and remove pharyngeal tissues (Fig. 1).
2. Cut the tissues into 3x3 mm fragments.
1. Place one fragment per well of 24-well tissue culture plates containing
1 ml TTCM/well.
2. Seal the plate and keep at 15°C in normal atmosphere.
Maintenance
of animals
Dissection
Culture
set-up