After selecting the optimal time and concentration
of melatonin treatments (0.1 mM, 1 mM melatonin for
72 hours), we performed the trypan blue dye exclusion
assay to measure cell viability. Treated and control P19
cells were trypsinized and washed with PBS. 100 μl of cell
suspension was aseptically transferred to a 1.5 ml tube and
an equal volume of trypan blue (T8154, Sigma) was added
for 3 minutes at room temperature. The resuspension
was then placed in a dual-use counting slide (145–0011,
Bio-Rad; Hercules, CA, USA) and read in a TC20
automated cell counter (145–0102SP, Bio-Rad). Results
are shown as percentage of live cells.