Composition of Salt Solutions and Culture Media
405
thionine 0.03, L-isoleucine 2.0, L-Ieucine 2.9, L-phenylalanine 1.B, diphosphopyridine
nucleotide 7.0, triphosphopyridine nucleotide 2.0, coenzyme A 2.5, cocarboxylase 2.0,
flavin-adenine dinucleotide 2.0, biotin 1.0, folic acid 1.0, choline chloride 1.0, nicotinamide 1.0, o-calcium pantothenate 1.0, pyridoxine hydrochloride 1.0, thiamine hydrochloride 1.0, riboflavin 0.1, inositol1.B, cyanocobalamin 10.0, nicotinic acid 2.0,paminobenzoic acid 2.0, pyridoxine hydrochloride 2.0, uridine 2.0, deoxyadenosine 10.0,
deoxycytidine 10.0, deoxyguanosine 10.0, thymidine 10.0, cytosine 1.0,
glucuronolactone 1.B, glutathione 10.0, phenol red 20.0, and glucose 100.0. The pH of
the solution should be adjusted to 7.2-7.4. The osmolarity of the solution is
155 mOsmol/kg. The medium is supplemented with 10% heat-inactivated FBS and 10%
whole egg ultrafiltrate. Sometimes hemolymph from adult Biomphalaria glabrata is
added at a concentration of 5% after inactivation at 60°C for 10 min. An antibiotic
mixture (penicillin G 100 IV, streptomycin sulfate 100 !lg, gentamicin sulfate 10 !lg/ml)
is added to the medium.
Botryllid cell culture medium (BCCM; Rinkevich and Rabinovitz, 1993)
The medium basically consists of Dulbecco's modified Eagle's medium x5 (DMEM)
and a salt solution, the osmolarity of which is adjusted to that of botryllid ascidian
hemolymph. The salt solution is made up of two major components at a 2x concentration.
Component A: NaCI13.67 g, KCI 0.412 g, and CaCl z ·2H z O 0.721 g, made up to 125 ml
with distilled water.
Component B: MgS04·7HzO 5.57 g and MgClz·6H z O 3.05 g, made up to 125 ml with
distilled water.
Both components are mixed (1 : 1), and the solution is sterilized by filtration through
a membrane filter with a 0.2 !lm pore size. Both stock solutions can be stored at 4°C.
The ready-to-use BCCM should be prepared before use. Each 100 ml BCCM is made
by mixing 20 ml DMEM (x5), 50 ml salt solution, 26 ml distilled water, 2 ml L-glutamine
solution (200 mM),2 ml HEPES buffer (1 M,pH 7.0) and 1 ml antibiotic mixture (penicillin sodium salt 10 4 IU, streptomycin 10 mg, and nystatin 1,250 IU). The medium is
sterilized by filtration through a membrane filter with a 0.2 !lm pore size, and can be
stored at 4°C for up to 3 weeks. Heat-inaysate (5%) 10.0 ml, and yeast extract (10%)
5.0 ml.
Cecil's medium modified by Heacox et al. for Nereis cell culture (Heacox et al., 1983)
Distilled water 147 ml, NaCI 6,390 mg, NaHC0 3 1,000 mg, KCI 340 mg, CaCl z ·2H z O
169 mg,glucose 1,000 mg,galactose 500 mg,fucose(o) 250 mg,fucose(L) 250 mg,minimal essential medium (MEM) essential amino acids (lOOx) 20.0 ml, MEM non-essential amino acids (100x) IB.O ml, MEM vitamin (100x) 3.3 ml, glutamine 200 mg, gentamicin 1.0 mg, penicillin 300,000 IV, Nereis coelomic fluid ultrafiltrate 0-20 ml, horse
serum 0-100 ml, hens' whole egg ultrafiltrate 0-100 ml, and Nereis vitellin 0 or 500 mg.
Chernin's nutrient medium (Chernin, 1963)
Chernin's BSS 850.0 ml, bovine amniotic fluid 110.0 ml, beef embryo extract 12.5 ml,
horse serum 12.5 ml, lactalbumin hydrolysate (5%) 10.0 ml, and yeast extract (10%)
5.0 ml.
405
thionine 0.03, L-isoleucine 2.0, L-Ieucine 2.9, L-phenylalanine 1.B, diphosphopyridine
nucleotide 7.0, triphosphopyridine nucleotide 2.0, coenzyme A 2.5, cocarboxylase 2.0,
flavin-adenine dinucleotide 2.0, biotin 1.0, folic acid 1.0, choline chloride 1.0, nicotinamide 1.0, o-calcium pantothenate 1.0, pyridoxine hydrochloride 1.0, thiamine hydrochloride 1.0, riboflavin 0.1, inositol1.B, cyanocobalamin 10.0, nicotinic acid 2.0,paminobenzoic acid 2.0, pyridoxine hydrochloride 2.0, uridine 2.0, deoxyadenosine 10.0,
deoxycytidine 10.0, deoxyguanosine 10.0, thymidine 10.0, cytosine 1.0,
glucuronolactone 1.B, glutathione 10.0, phenol red 20.0, and glucose 100.0. The pH of
the solution should be adjusted to 7.2-7.4. The osmolarity of the solution is
155 mOsmol/kg. The medium is supplemented with 10% heat-inactivated FBS and 10%
whole egg ultrafiltrate. Sometimes hemolymph from adult Biomphalaria glabrata is
added at a concentration of 5% after inactivation at 60°C for 10 min. An antibiotic
mixture (penicillin G 100 IV, streptomycin sulfate 100 !lg, gentamicin sulfate 10 !lg/ml)
is added to the medium.
Botryllid cell culture medium (BCCM; Rinkevich and Rabinovitz, 1993)
The medium basically consists of Dulbecco's modified Eagle's medium x5 (DMEM)
and a salt solution, the osmolarity of which is adjusted to that of botryllid ascidian
hemolymph. The salt solution is made up of two major components at a 2x concentration.
Component A: NaCI13.67 g, KCI 0.412 g, and CaCl z ·2H z O 0.721 g, made up to 125 ml
with distilled water.
Component B: MgS04·7HzO 5.57 g and MgClz·6H z O 3.05 g, made up to 125 ml with
distilled water.
Both components are mixed (1 : 1), and the solution is sterilized by filtration through
a membrane filter with a 0.2 !lm pore size. Both stock solutions can be stored at 4°C.
The ready-to-use BCCM should be prepared before use. Each 100 ml BCCM is made
by mixing 20 ml DMEM (x5), 50 ml salt solution, 26 ml distilled water, 2 ml L-glutamine
solution (200 mM),2 ml HEPES buffer (1 M,pH 7.0) and 1 ml antibiotic mixture (penicillin sodium salt 10 4 IU, streptomycin 10 mg, and nystatin 1,250 IU). The medium is
sterilized by filtration through a membrane filter with a 0.2 !lm pore size, and can be
stored at 4°C for up to 3 weeks. Heat-inaysate (5%) 10.0 ml, and yeast extract (10%)
5.0 ml.
Cecil's medium modified by Heacox et al. for Nereis cell culture (Heacox et al., 1983)
Distilled water 147 ml, NaCI 6,390 mg, NaHC0 3 1,000 mg, KCI 340 mg, CaCl z ·2H z O
169 mg,glucose 1,000 mg,galactose 500 mg,fucose(o) 250 mg,fucose(L) 250 mg,minimal essential medium (MEM) essential amino acids (lOOx) 20.0 ml, MEM non-essential amino acids (100x) IB.O ml, MEM vitamin (100x) 3.3 ml, glutamine 200 mg, gentamicin 1.0 mg, penicillin 300,000 IV, Nereis coelomic fluid ultrafiltrate 0-20 ml, horse
serum 0-100 ml, hens' whole egg ultrafiltrate 0-100 ml, and Nereis vitellin 0 or 500 mg.
Chernin's nutrient medium (Chernin, 1963)
Chernin's BSS 850.0 ml, bovine amniotic fluid 110.0 ml, beef embryo extract 12.5 ml,
horse serum 12.5 ml, lactalbumin hydrolysate (5%) 10.0 ml, and yeast extract (10%)
5.0 ml.