158
Methods for Setting Up Primary Cultures Specific to Animal Groups
Chemicals - Paraffin
- Petrolatum
Animals - Starfish Patiria miniata or Pisaster ochraceus segnis (the animals are
maintained in the laboratory at 16.5° or 19°C by means of a water bath
or a recirculating seawater system)
• • Procedure
Bleeding l. Withdraw coelomic fluid using a syringe, the inner surface of which
has been coated with silicone. Insert the needle through one of the
skeletal openings for the tube feet, which occur along the ambulacral
groove, or through one of the dermal papulae of the aboral surface.
2. Dispense the coelomic fluid into test tubes, and keep the tubes in a
refrigerator at 4°C.
3. Remove clots of coelomic cells, which form during storage, from the
test tube using a capillary pipette.
Culture l. Place the coelomic cell clot and attendant fluid on a cover glass.
set-up 2. Invert the cover glass over the well of a depression slide, and seal the
edge of the cover glass with melted paraffin and petrolatum.
3. Maintain the hanging drop cultures at 4°C.
Results
According to Johnson and Beeson (1966), the clots in the hanging drop
cultures were maintained in good condition for 3 weeks at 4°C. Only one
type of coelomocyte occurred regularly in Patiria cultures, and this was
a phagocytic amoebocyte. Revolving cysts with flagella appeared in the
hanging drops, but the origin and function of these cysts were obscure.
Subprotocol 4
Coelomocytes from Sea Urchins (Echinoidea)
The following procedure is based on the methods of Johnson (1969) .
• • Materials
Equipment - Plastic bags
- Syringes with 3.18 cm, 22-G hypodermic needles
- 15-ml conical-bottom centrifuge tubes
- Pasteur pipettes
- Cover glasses
- Depression slide glasses
Miscellaneous - Bees wax
Methods for Setting Up Primary Cultures Specific to Animal Groups
Chemicals - Paraffin
- Petrolatum
Animals - Starfish Patiria miniata or Pisaster ochraceus segnis (the animals are
maintained in the laboratory at 16.5° or 19°C by means of a water bath
or a recirculating seawater system)
• • Procedure
Bleeding l. Withdraw coelomic fluid using a syringe, the inner surface of which
has been coated with silicone. Insert the needle through one of the
skeletal openings for the tube feet, which occur along the ambulacral
groove, or through one of the dermal papulae of the aboral surface.
2. Dispense the coelomic fluid into test tubes, and keep the tubes in a
refrigerator at 4°C.
3. Remove clots of coelomic cells, which form during storage, from the
test tube using a capillary pipette.
Culture l. Place the coelomic cell clot and attendant fluid on a cover glass.
set-up 2. Invert the cover glass over the well of a depression slide, and seal the
edge of the cover glass with melted paraffin and petrolatum.
3. Maintain the hanging drop cultures at 4°C.
Results
According to Johnson and Beeson (1966), the clots in the hanging drop
cultures were maintained in good condition for 3 weeks at 4°C. Only one
type of coelomocyte occurred regularly in Patiria cultures, and this was
a phagocytic amoebocyte. Revolving cysts with flagella appeared in the
hanging drops, but the origin and function of these cysts were obscure.
Subprotocol 4
Coelomocytes from Sea Urchins (Echinoidea)
The following procedure is based on the methods of Johnson (1969) .
• • Materials
Equipment - Plastic bags
- Syringes with 3.18 cm, 22-G hypodermic needles
- 15-ml conical-bottom centrifuge tubes
- Pasteur pipettes
- Cover glasses
- Depression slide glasses
Miscellaneous - Bees wax