198
Methods for Setting Up Primary Cultures Specific to Animal Groups
Separation
of cell types
Culture
set-up
1. Wash the cells four times in NBSS. Allow the cells to settle, and pipette
off the supernatant between washes. Eleocytes are smaller than large
oocytes, and settle above the oocytes during washing. Pipette off the
eleocyte layer, leaving an oocyte preparation.
2. Place the cells on a sterile 75-j.1m mesh nylon net and wash them gently with sterile NBSS (see Comments).
3. Transfer the collected oocytes into sterile NBSS containing antibiotic
mixture 2 for 30 min.
1. Collect the oocytes and suspend them in culture medium.
2. Dispense the cell suspension to culture vessels to a cell density of 5001,000 cells/ml.
Comments
Density gradient: medium-sized oocytes (average diameter 60-120 j.1m)
can be separated from eleocytes on sterile discontinuous Ficoll gradients, which range from 2% to 20% in NBSS at steps of 1 % or 2%. The
coelomic cells are placed on the gradient and centrifuged for 5 min at
160 g at 4°C. The green oocytes are seen easily and can be removed from
the gradient with a Pasteur pipette. Oocytes are then placed on a 35-j.1m
mesh nylon net for washing.
Results
Oocytes do not divide, but they increase in size. This type of growth
continues for up to 4 weeks.
Subprotocol 2
Neurons of Leeches (Hirudinea)
Although nerve cells do not proliferate in vitro, the long-term survival
and formation of neurons have been reported by Dietzel et al. (1986).
Materials
Equipment - Inverted microscope
- Fire-polished micropipette
- Pasteur pipette
- Petri dish
- Polylysine-coated microtest culture dish (no. 3008; Falcon, BectonDickinson)
Solutions - Culture medium consisting of Leibovitz's L-15 medium (Gibco) supplemented with gentamicin (0.1 mg/ml), glucose (6 mg/ml), and a mixture of enzymes (collagenase and dispase; 2 mg/ml; BoehringerMannheim)
Methods for Setting Up Primary Cultures Specific to Animal Groups
Separation
of cell types
Culture
set-up
1. Wash the cells four times in NBSS. Allow the cells to settle, and pipette
off the supernatant between washes. Eleocytes are smaller than large
oocytes, and settle above the oocytes during washing. Pipette off the
eleocyte layer, leaving an oocyte preparation.
2. Place the cells on a sterile 75-j.1m mesh nylon net and wash them gently with sterile NBSS (see Comments).
3. Transfer the collected oocytes into sterile NBSS containing antibiotic
mixture 2 for 30 min.
1. Collect the oocytes and suspend them in culture medium.
2. Dispense the cell suspension to culture vessels to a cell density of 5001,000 cells/ml.
Comments
Density gradient: medium-sized oocytes (average diameter 60-120 j.1m)
can be separated from eleocytes on sterile discontinuous Ficoll gradients, which range from 2% to 20% in NBSS at steps of 1 % or 2%. The
coelomic cells are placed on the gradient and centrifuged for 5 min at
160 g at 4°C. The green oocytes are seen easily and can be removed from
the gradient with a Pasteur pipette. Oocytes are then placed on a 35-j.1m
mesh nylon net for washing.
Results
Oocytes do not divide, but they increase in size. This type of growth
continues for up to 4 weeks.
Subprotocol 2
Neurons of Leeches (Hirudinea)
Although nerve cells do not proliferate in vitro, the long-term survival
and formation of neurons have been reported by Dietzel et al. (1986).
Materials
Equipment - Inverted microscope
- Fire-polished micropipette
- Pasteur pipette
- Petri dish
- Polylysine-coated microtest culture dish (no. 3008; Falcon, BectonDickinson)
Solutions - Culture medium consisting of Leibovitz's L-15 medium (Gibco) supplemented with gentamicin (0.1 mg/ml), glucose (6 mg/ml), and a mixture of enzymes (collagenase and dispase; 2 mg/ml; BoehringerMannheim)