5.1.1 Primary cell cultures from the caudal fin †
220.127.116.11 Materials †
- Two to three of healthy young adult medaka,
- Sodium hypochlorite solution (Wako, Osaka, Japan)
- Gelatin-coated plastic wells: wells of four-well plates (Nunc, Roskilde, -Denmark) treated with the 0.1% gelatin solution
18.104.22.168 Methods †
- Cut off the posterior half of the caudal fin of two-to three young adult fish.
- Immerse the tissues in the phosphate buffered saline (PBS) containing 0.4% of sodium hypochlorite for 30 seconds.
- Rinse the tissues by three changes of PBS.
- Mince the tissues into small fragments in a small amount of PBS.
- Remove the PBS and add the culture medium, Leibovitz’s L-15 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 20% fetal bovine serum (FBS; Gibco-BRL), 2 mML-glutamine, 100 U/mL penicillin and 100µg/mL streptomycin..
- Rince the tissue fragments by three changes of the culture medium.
- The tissue fragments are then inoculated in a gelatin-coated well containing 0.5 mL of the culture medium.
- The culture is incubated at 28C in air. Do not disturb the culture for several days after inoculation until the tissue fragments attach the well.
- Then a part of the culture medium is changed every 3 to 4 days, depending on the extent of cell outgrowth from tissue fragments.