Hoechst 33258 staining assay

Jurkat cells were collected and resuspended to adjust the cell density to 2x10⁵ cells/ml. The suspension was added to a 6-well plate at 2 ml/well. Following the addition of different concentrations (0, 3, 6, and 9 µM) of hirsutanol A, the cells were incubated for 48 h. Subsequently, the cells were collected, Hoechst 33258 working solution (5 µg/ml) was added to fully cover the cells and the cells were then placed in an incubator at 37˚C for 30 min. The staining solution was discarded via centrifugation (300 x g at room temperature for 5 min) and the cells were washed with PBS twice, followed by observation via fluorescence microscopy (maximum excitation wavelength, 352 mm; maximum emission wavelength, 461 mm). Each sample was viewed from five fields with roughly the same number of cells.