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Methods for Setting Up Primary Cultures Specific to Animal Groups
Subprotocol 3
Embryos of the Colonial Protochordates (Tunicata)
Materials
Equipment - Dissecting microscope
- Centrifuge and IO-ml plastic screw-capped centrifuge tubes
- I.S-ml micro centrifuge tubes
- Soft brush such as paintbrush
- Syringe with a 28 G needle
- Pasteur pipette
- Sterile plastic pestle
- 24-Well flat-bottom tissue culture plate (Nunc)
Solutions - Washing salt solution (WS; see Appendix 1)
- BCCM (see Appendix 1)
Animals - Colonies of colonial proto chordates Botryllus schlosseri (see instructions given for subprotocol 2 animals)
Procedure
Sterilization 1. Clean the animals carefully with a soft brush.
2. Wipe the animals attached to glass slides with 70% ethanol.
3. Transfer the animals to a glass dish containing Millipore-filtered seawater at room temperature.
Dissection 1. Make a longitudinal incision to the dorsal tissues between both siphons of selected zooids under a dissecting microscope with a sterile
syringe equipped with a 28 G needle (cf. Fig. 1).
2. Free the embryos carefully from the atrial chambers using the needle
tip.
3. Collect the freed embryos with sterile Pasteur pipettes, and transfer
them to a lO-ml plastic screw-capped centrifuge tube.
4. Rinse the embryos at least 10 times with WS by centrifugation at 1 g
for 1 min.
5. Suspend the embryos in BCCM less than 1.5 ml.
6. Transfer the suspension to a I.S-ml micro centrifuge tube.
Dissociation 1. Crush the embryos by gentle pressure with a sterile plastic pestle (see
Comments).
Culture 1. Transfer the resulting cell suspension to a IS-ml centrifuge tube.
set-up 2. Add BCCM so as to make the cell density 10 5 cells/ml.
3. Dispense 0.5 ml cell suspension to each well of a 24-well plate.
Methods for Setting Up Primary Cultures Specific to Animal Groups
Subprotocol 3
Embryos of the Colonial Protochordates (Tunicata)
Materials
Equipment - Dissecting microscope
- Centrifuge and IO-ml plastic screw-capped centrifuge tubes
- I.S-ml micro centrifuge tubes
- Soft brush such as paintbrush
- Syringe with a 28 G needle
- Pasteur pipette
- Sterile plastic pestle
- 24-Well flat-bottom tissue culture plate (Nunc)
Solutions - Washing salt solution (WS; see Appendix 1)
- BCCM (see Appendix 1)
Animals - Colonies of colonial proto chordates Botryllus schlosseri (see instructions given for subprotocol 2 animals)
Procedure
Sterilization 1. Clean the animals carefully with a soft brush.
2. Wipe the animals attached to glass slides with 70% ethanol.
3. Transfer the animals to a glass dish containing Millipore-filtered seawater at room temperature.
Dissection 1. Make a longitudinal incision to the dorsal tissues between both siphons of selected zooids under a dissecting microscope with a sterile
syringe equipped with a 28 G needle (cf. Fig. 1).
2. Free the embryos carefully from the atrial chambers using the needle
tip.
3. Collect the freed embryos with sterile Pasteur pipettes, and transfer
them to a lO-ml plastic screw-capped centrifuge tube.
4. Rinse the embryos at least 10 times with WS by centrifugation at 1 g
for 1 min.
5. Suspend the embryos in BCCM less than 1.5 ml.
6. Transfer the suspension to a I.S-ml micro centrifuge tube.
Dissociation 1. Crush the embryos by gentle pressure with a sterile plastic pestle (see
Comments).
Culture 1. Transfer the resulting cell suspension to a IS-ml centrifuge tube.
set-up 2. Add BCCM so as to make the cell density 10 5 cells/ml.
3. Dispense 0.5 ml cell suspension to each well of a 24-well plate.