154
Methods for Setting Up Primary Cultures Specific to Animal Groups
Embryo
collection
Dissociation
op
Fig. 1. Inner structure of Asterias rubens. One arm has been left intact, the hepatic
caeca and gonads have been cut away from two arms, and, from one of these, the
ampullae of the tube feet and retractor muscles of the cardiac stomach have also
been removed. a, anus; amb.oss, ambulacral ossicle; amb.p, ambulacral pore; amp,
ampulla of tube foot; card.st, cardiac stomach; mad, madreporite; op, optic cushion; ov, ovary; pyl.cae, pyloric caecum; pyl.st, pyloric stomach; r.s, rectal sac; ret.mu,
retractor muscle of cardiac stomach; sept, interradial septum. From Fretter, V. and
Graham, A., "A Functional Anatomy ofInvertebrates", Fig.149. Copyright © 1976 by
Academic Press, Inc. Reproduced with permission of copyright owner
lUI Procedure
1. Collect packed embryos (0.2 ml) at the mesenchyme migration stage
(approximately 28-30 h after fertilization) using a hand centrifuge.
2. Wash the packed embryos three times with 10 ml SSw.
1. Suspend the embryos in 4.0 ml dissociation medium.
2. Leave for 5 min.
3. Apply 10 strokes of pipetting with a Pasteur pipette.
4. Hand centrifuge the resulting cell suspension in order to separate single
cells.
5. Set the dissociated single cells aside in a separate centrifuge tube while
the pellet is dissociated again in the same manner.
6. Pool the obtained single cell suspension.
7. Centrifuge this cell suspension at 250 g for 5 min.
Methods for Setting Up Primary Cultures Specific to Animal Groups
Embryo
collection
Dissociation
op
Fig. 1. Inner structure of Asterias rubens. One arm has been left intact, the hepatic
caeca and gonads have been cut away from two arms, and, from one of these, the
ampullae of the tube feet and retractor muscles of the cardiac stomach have also
been removed. a, anus; amb.oss, ambulacral ossicle; amb.p, ambulacral pore; amp,
ampulla of tube foot; card.st, cardiac stomach; mad, madreporite; op, optic cushion; ov, ovary; pyl.cae, pyloric caecum; pyl.st, pyloric stomach; r.s, rectal sac; ret.mu,
retractor muscle of cardiac stomach; sept, interradial septum. From Fretter, V. and
Graham, A., "A Functional Anatomy ofInvertebrates", Fig.149. Copyright © 1976 by
Academic Press, Inc. Reproduced with permission of copyright owner
lUI Procedure
1. Collect packed embryos (0.2 ml) at the mesenchyme migration stage
(approximately 28-30 h after fertilization) using a hand centrifuge.
2. Wash the packed embryos three times with 10 ml SSw.
1. Suspend the embryos in 4.0 ml dissociation medium.
2. Leave for 5 min.
3. Apply 10 strokes of pipetting with a Pasteur pipette.
4. Hand centrifuge the resulting cell suspension in order to separate single
cells.
5. Set the dissociated single cells aside in a separate centrifuge tube while
the pellet is dissociated again in the same manner.
6. Pool the obtained single cell suspension.
7. Centrifuge this cell suspension at 250 g for 5 min.