5.1.1 primary cell culture from the caudal fin

FrontPage

5.1.1 Primary cell cultures from the caudal fin†

Yuko Wakamatsu
Nagoya University

5.1.1.0 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

5.1.1.1 Methods†

1. Cut off the posterior half of the caudal fin of two-to three young adult fish.
2. Immerse the tissues in the phosphate buffered saline (PBS) containing 0.4% of sodium hypochlorite for 30 seconds.
3. Rinse the tissues by three changes of PBS.
4. Mince the tissues into small fragments in a small amount of PBS.
5. 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..
6. Rince the tissue fragments by three changes of the culture medium.
7. The tissue fragments are then inoculated in a gelatin-coated well containing 0.5 mL of the culture medium.
8. 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.
9. Then a part of the culture medium is changed every 3 to 4 days, depending on the extent of cell outgrowth from tissue fragments.