Hoechst 33258 staining of nuclei for analysis of apoptotic cells

Hoechst 33258 staining of nuclei was used to detect apoptotic cells. Hoechst 33258, a blue fluorescent dye, readily permeates the cell membrane for the determination and semi-quantification of cells with fragmented and condensed chromatin. Following the 24 h re-oxygenation period, the NSCs were washed three times with PBS and fixed in 4% formaldehyde for 10 min. The fixed cells were then washed with PBS and incubated with Hoechst 33258 (Sigma-Aldrich) for 5 min in a humidified chamber, protected from light, at 37°C. The cells were washed three times with PBS and mounted with one drop of mounting solution. The cell nuclei were observed and images were captured using a fluorescence microscope (Olympus BX51; Olympus Corporation, Tokyo, Japan).