Characterization of SP cells by flow cytometry.

(A) FACS analysis on single cell suspension of human NSCLC cell lines stained with Hoechst 33342 dye showing SP cells. SP cells are enclosed within the area demarcated in pink. Fumitremorgin C inhibited the efflux of the dye and caused the disappearance of SP cells. The frequency of SP cells is represented. (B, C) Cell cycle analysis using area-histogram parameter for the blue emission of Hoechst 33342 as described in the text. The profile shown in B is the representative for all the four cell lines. (D) Aldefluor assay on Hoechst 33342 stained H1975 and H1650 cells. The base line fluorescence was established by inhibiting ALDH activity with DEAB (Left) to generate the gate to identify ALDH-Hi cells in total as well as the SP and MP cells that have not been incubated with DEAB (three right panels). All ALDH-Hi cells are enclosed within the area demarcated in pink. The fold difference in frequency of ALDH-Hi cells between SP and MP cells is plotted.