132
Methods for Setting Up Primary Cultures Specific to Animal Groups
tion occurred from blind fragments ofhepatopancreas tubules, and some
cultures were subcultured for 17 and 28 generations over a period of
more than 5 months in vitro.
Comments
Ghosh et al. (1995) cultured prawn hepatocytes obtained by the injection of heparin and perfusion of the hepatopancreas in serum-free modified TC-199 medium. The cultures were maintained at 15° and 23°C, but
the cells were short lived.
Subprotocol 7
Shrimp (Crustacea) Lymphoid Organs (Oka Organs) and
Ovaries
The following protocol is based on the methods of Chen et al. (1989).
Materials
Equipment - Centrifuge and centrifuge tubes
- Petri dish
- Dissecting tray
- Forceps
- Scissors
- Pasteur pipette
- 25-cmz Plastic flask (Falcon, Becton-Dickinson)
- 6-Well tissue culture plate (Primaria; Falcon, Becton-Dickinson)
- 15-ml Syringe with a no. 19 needle
Solutions - Double-strength Leibovitz's L-15 medium (see Appendix 1)
- Shrimp tissue culture medium (STCM)
- Double-strength L-15 medium 70%
- FBS 5%
- Shrimp extract (see Comments) 8%-10%
- Lobster hemolymph (see Comments) 15%
- Streptomycin 100 Ilg/ml
- Penicillin 100 IV/ml
- The osmolarity of the medium is adjusted to 720 mOsmollkg with
the following salt mixture
- Salt mixture
- Dissolve 102,4 gNaCl, 1.8 g KCI,5.1 g CaCl z , 10.8 g MgS0 4 ,and 11.8 g
MgCl z in 1,000 ml distilled water
Animals - Adult shrimp (Penaeus penicillatus), weighing approximately 40 g, or
Penaeus stylirostris or Penaeus vannamei
Methods for Setting Up Primary Cultures Specific to Animal Groups
tion occurred from blind fragments ofhepatopancreas tubules, and some
cultures were subcultured for 17 and 28 generations over a period of
more than 5 months in vitro.
Comments
Ghosh et al. (1995) cultured prawn hepatocytes obtained by the injection of heparin and perfusion of the hepatopancreas in serum-free modified TC-199 medium. The cultures were maintained at 15° and 23°C, but
the cells were short lived.
Subprotocol 7
Shrimp (Crustacea) Lymphoid Organs (Oka Organs) and
Ovaries
The following protocol is based on the methods of Chen et al. (1989).
Materials
Equipment - Centrifuge and centrifuge tubes
- Petri dish
- Dissecting tray
- Forceps
- Scissors
- Pasteur pipette
- 25-cmz Plastic flask (Falcon, Becton-Dickinson)
- 6-Well tissue culture plate (Primaria; Falcon, Becton-Dickinson)
- 15-ml Syringe with a no. 19 needle
Solutions - Double-strength Leibovitz's L-15 medium (see Appendix 1)
- Shrimp tissue culture medium (STCM)
- Double-strength L-15 medium 70%
- FBS 5%
- Shrimp extract (see Comments) 8%-10%
- Lobster hemolymph (see Comments) 15%
- Streptomycin 100 Ilg/ml
- Penicillin 100 IV/ml
- The osmolarity of the medium is adjusted to 720 mOsmollkg with
the following salt mixture
- Salt mixture
- Dissolve 102,4 gNaCl, 1.8 g KCI,5.1 g CaCl z , 10.8 g MgS0 4 ,and 11.8 g
MgCl z in 1,000 ml distilled water
Animals - Adult shrimp (Penaeus penicillatus), weighing approximately 40 g, or
Penaeus stylirostris or Penaeus vannamei