13 Prochordata
145
by severing the stolons immediately posterior to the body cavity
{Fig. 2)
- Centrifuge the hemolymph at 1,000 g for 20 min at 4°C
- Store the supernatant at -20°C until use (the supernatant can be
stored for up to 3 months)
Results
In cultures of pharyngeal tissues from S. clava, Raftos et al. (1990) found
that the pharyngeal explants survived for more than 72 days. Contamination with protistans occurred at a high frequency (26%). The predominant contaminating protistans were ciliates (Prostomata and
Litostomatea). The contamination usually became evident after 20 days'
culture. Muscular contraction of the explants lasted up to 44 days. Ciliated epithelial cells showed ciliary activity for more than 2 months. Total
hemocytes in the sinuses of the explants decreased during culture. This
loss of hemocytes from the explants is partly due to an efflux of cells into
the culture medium. The migrated hemocytes survived throughout the
culture period. Cell proliferation was observed in the explants.
Subprotocol 2
Circulatory Hemocytes of Colonial Protochordates (Tunicata)
Long-term cultures of freely circulating hemocytes have become possible for colonial proto chordates. The following procedure is the method
of Rinkevich and Rabinowitz (1993) for the cultivation of circulatory
hemocytes from Botryllus schlosseri.
Materials
- Aquarium or water tank
- Aerating system
- Airstone for commercial aerator
- Single-edged surgical razor blades (no. 1; VWR)
- Petri dish (9 cm)
- Forceps
- Nylon mesh (3D mm)
- Centrifuge and centrifuge tubes
- Soft brush such as paintbrush
- 24-Well tissue culture plate
- Settlement glass slide (5 x 7.5 cm)
- Buffered washing solution (WS; see Appendix 1)
- Botryllid cell culture medium (BCCM; see Appendix 1)
- Seawater (natural)
Equipment
Solutions
145
by severing the stolons immediately posterior to the body cavity
{Fig. 2)
- Centrifuge the hemolymph at 1,000 g for 20 min at 4°C
- Store the supernatant at -20°C until use (the supernatant can be
stored for up to 3 months)
Results
In cultures of pharyngeal tissues from S. clava, Raftos et al. (1990) found
that the pharyngeal explants survived for more than 72 days. Contamination with protistans occurred at a high frequency (26%). The predominant contaminating protistans were ciliates (Prostomata and
Litostomatea). The contamination usually became evident after 20 days'
culture. Muscular contraction of the explants lasted up to 44 days. Ciliated epithelial cells showed ciliary activity for more than 2 months. Total
hemocytes in the sinuses of the explants decreased during culture. This
loss of hemocytes from the explants is partly due to an efflux of cells into
the culture medium. The migrated hemocytes survived throughout the
culture period. Cell proliferation was observed in the explants.
Subprotocol 2
Circulatory Hemocytes of Colonial Protochordates (Tunicata)
Long-term cultures of freely circulating hemocytes have become possible for colonial proto chordates. The following procedure is the method
of Rinkevich and Rabinowitz (1993) for the cultivation of circulatory
hemocytes from Botryllus schlosseri.
Materials
- Aquarium or water tank
- Aerating system
- Airstone for commercial aerator
- Single-edged surgical razor blades (no. 1; VWR)
- Petri dish (9 cm)
- Forceps
- Nylon mesh (3D mm)
- Centrifuge and centrifuge tubes
- Soft brush such as paintbrush
- 24-Well tissue culture plate
- Settlement glass slide (5 x 7.5 cm)
- Buffered washing solution (WS; see Appendix 1)
- Botryllid cell culture medium (BCCM; see Appendix 1)
- Seawater (natural)
Equipment
Solutions