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Methods for Setting Up Primary Cultures Specific to Animal Groups
- Centrifuge and centrifuge tube
- Round- or flat-bottomed tissue culture plates
Solutions - Culture medium (the stock solution is Leibovitz's L-15 medium supplemented with 1.64 gIl CaCI 2 ·2H 2 0, 0.52 gIl KCI, 6.07 gIl MgS04·7H20,
21.2 gIl HEPES (20 mllI), and 0.04 gIl taurine; the final culture medium consists of 10%-95% stock solution, 5% or 10% heat -inactivated
FBS, and sterile seawater (SSW) to make 100 %; antibiotics (0.5 mgl
ml gentamicin sulfate, 10 2 IU/ml sodium penicillin, 0.1 mglml streptomycin, 12.5 IU/ml amphotericin, or 12.5 IU/ml nystatin) are added
to the medium for primary culture)
- Freezing medium (a mixture of phosphate-buffered saline, adjusted
with NaCI to the osmolarity of seawater, and DMSO in a ratio of 8: 2,
or a mixture of L-15 medium, heat-inactivated FBS, and glycerol in a
ratio of 8 : 1 : 1)
- Self-generating continuous density gradient (place 6.54 ml Percoll
stock solution (density 1.13 g/ml) into a lO-ml centrifuge tube; add
3.46 ml double-distilled water, and adjust the osmolarity of the solution to that of seawater by adding 0.29 g NaCl. Centrifuge at 20,000 g
at 4°C for 1 h)
Chemicals - Percoll
Animals - Colonial cnidarians (collect a part of colonies from the sea, and rinse
with SSW (filtered through a membrane filter with a 0.22 /lm pore
size); keep the animals in SSW supplemented with antibiotics (e.g. 1 %
gentamicin sulphate) at 4°C for up to 24 h before dissociation of cells)
Procedure
Following is the spontaneous dissociation method developed by Frank
et al. (1994).
Dissociation 1. Take small fragments (0.5-1.0 em) from the collected colonial
cnidarian.
2. Rinse the fragments in SSW.
3. Transfer the fragments into freezing medium.
4. Cool the fragments gradually to the temperature ofliquid nitrogen (196°C).
5. Thaw the fragments at room temperature.
6. Transfer the fragments into culture medium. Dissociation of the tissue will occur spontaneously within 10 days.
Cell 1. Rinse the cell suspension in SSW.
separation 2. Stain the cells with 0.001 % neutral red.
3. Load the cell suspension on the surface of the Percoll gradient (up to
2 X 10 7 cells in 2 ml SSW).
Methods for Setting Up Primary Cultures Specific to Animal Groups
- Centrifuge and centrifuge tube
- Round- or flat-bottomed tissue culture plates
Solutions - Culture medium (the stock solution is Leibovitz's L-15 medium supplemented with 1.64 gIl CaCI 2 ·2H 2 0, 0.52 gIl KCI, 6.07 gIl MgS04·7H20,
21.2 gIl HEPES (20 mllI), and 0.04 gIl taurine; the final culture medium consists of 10%-95% stock solution, 5% or 10% heat -inactivated
FBS, and sterile seawater (SSW) to make 100 %; antibiotics (0.5 mgl
ml gentamicin sulfate, 10 2 IU/ml sodium penicillin, 0.1 mglml streptomycin, 12.5 IU/ml amphotericin, or 12.5 IU/ml nystatin) are added
to the medium for primary culture)
- Freezing medium (a mixture of phosphate-buffered saline, adjusted
with NaCI to the osmolarity of seawater, and DMSO in a ratio of 8: 2,
or a mixture of L-15 medium, heat-inactivated FBS, and glycerol in a
ratio of 8 : 1 : 1)
- Self-generating continuous density gradient (place 6.54 ml Percoll
stock solution (density 1.13 g/ml) into a lO-ml centrifuge tube; add
3.46 ml double-distilled water, and adjust the osmolarity of the solution to that of seawater by adding 0.29 g NaCl. Centrifuge at 20,000 g
at 4°C for 1 h)
Chemicals - Percoll
Animals - Colonial cnidarians (collect a part of colonies from the sea, and rinse
with SSW (filtered through a membrane filter with a 0.22 /lm pore
size); keep the animals in SSW supplemented with antibiotics (e.g. 1 %
gentamicin sulphate) at 4°C for up to 24 h before dissociation of cells)
Procedure
Following is the spontaneous dissociation method developed by Frank
et al. (1994).
Dissociation 1. Take small fragments (0.5-1.0 em) from the collected colonial
cnidarian.
2. Rinse the fragments in SSW.
3. Transfer the fragments into freezing medium.
4. Cool the fragments gradually to the temperature ofliquid nitrogen (196°C).
5. Thaw the fragments at room temperature.
6. Transfer the fragments into culture medium. Dissociation of the tissue will occur spontaneously within 10 days.
Cell 1. Rinse the cell suspension in SSW.
separation 2. Stain the cells with 0.001 % neutral red.
3. Load the cell suspension on the surface of the Percoll gradient (up to
2 X 10 7 cells in 2 ml SSW).