2.6. Detection of Apoptosis (TUNEL Assay)

The terminal transferase dUTP nick end labeling (TUNEL) assay kit was purchased from Roche Diagnostic Corporation. For detection of apoptosis, TUNEL assay was performed following the manufacturer’s instructions. Briefly, the fixation process was performed as described above. After fixation, the samples were permeabilized with 0.1% Triton X-100 in PBS for 30 min at 37 °C. The TUNEL reaction was then performed using the manufacturer’s instructions. Hoechst33342 (5 mg mL⁻¹ in PBS, Gibco) was used to visualize the nuclei. The state of cell clusters stained by the TUNEL was detected by a Lecia DMI8 confocal laser scanning microscope. Images of five to six randomly chosen fields of vision (one-two spheroids per field) were taken.