24 well or 96 well plates

Japanese flounder head kidney primary cells were prepared as described previously.^{18 (link)} Japanese flounder HKMs and peripheral blood leukocytes (PBLs) were
isolated by discontinuous Percoll density (1.020/1.070 and 1.070/1.077,
respectively, GE Biosciences) gradient centrifugation. After centrifugation at
3000 r/min for 40 min at 4°C, the white interface fraction was collected and
washed three times with cold PBS. Cell viability was examined by Trypan blue
exclusion test, which showed greater than 95% viability. Japanese flounder HKMs
and PBLs were then cultured in 24-well or 96-well plates (Corning) with RPMI
1640 medium supplemented with 10% FBS (Invitrogen) and 1%
penicillin–streptomycin liquid at 21°C overnight before experimentation.
P. olivaceus head kidney cells were seeded into 24-well
plates (ThermoFisher Scientific) and were cultured in DMEM-F12 medium
supplemented with 10% FBS, penicillin (100 IU/ml), and streptomycin (100 μg/ml)
at 21°C under 2.5% CO₂ atmosphere.

Mouse DRG neurons were dissected from embryonic days 13.5 or 14.5 from CD1 mouse embryos. DRG neurons were incubated with 0.05% trypsin containing 0.02% EDTA at 37°C for 20 min and then resuspended in neurobasal media (Gibco) containing 2% B27 (Invitrogen), 50 ng/ml nerve growth factor (Harlan Laboratories), 1 µM 5-fluoro-2deoxyuridine (Sigma-Aldrich), 1 µM uridine (Sigma-Aldrich), and penicillin/streptomycin (Thermo Fisher Scientific). The dissociated DRG neurons were plated in 24-well or 96-well plates (Corning) coated with poly-D-lysine (0.1 mg/ml; Sigma-Aldrich) and laminin (3 µg/ml; Invitrogen) and then allowed to adhere in humidified tissue culture incubator (5% CO₂) for 15 min. Finally, DRG growth medium was gently added (500 µl for 24-well, 100 µl for 96-well). Lentivirus was transduced at 1 or 2 d in vitro (DIV). At DIV7, assays for axon degeneration and/or cell death were performed by applying BB (or TNF-α) or axotomy.