Trypsin versene solution

The entire brain from CyHV-2 latently infected fish was collected and washed twice in Medium 199 containing streptomycin (100 µg/mL) and penicillin (100 U/mL). The washed brain was minced with scissors into small pieces and digested into a single cell suspension with 0.25% trypsin-versene solution (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 15 min. The digested cells were then placed in M199 medium containing 10% heat-inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA, USA) and filtered with a 70-µm steel mesh. The filtered cell suspensions were centrifuged at 200× g for 5 min and resuspended in M199 medium supplemented with 20% FBS containing streptomycin and penicillin antibiotics and cultured in 25 cm² tissue culture flasks (Corning, NY, USA) at 28 °C with 5% CO₂. Approximately 50% of the medium was replaced with fresh cell culture medium every 3 days. The confluent monolayer was split at a ratio of 1:2 every 6–8 days. After 15 subcultures, the cells were cultured in M199 medium with 10% FBS.

Cells at the exponential growth phase were harvested using trypsin-versene solution (Sigma) and seeded into 96-well plates at 2×10⁴ cells/well in 100 µL. Following overnight incubation and the formation of a cell monolayer, test cells were treated with Tumor necrosis factor-1 alpha (TNF-α) at selected concentrations. Seventy-two hours later, 20 µL of MTT solution (5 mg/mL) was added to the 100 µL of medium in each well, and the plate incubated at selected temperatures for 4 hour. The solution was removed followed by adding 100 µL/well of DMSO to solubilize the purple formazan crystals produced. Absorbance in each well was measured at 570 nm using a 96-well plate reader (Beckman Coulter, Fullerton, CA).