Identification and analysis of H1650 and H1650-ER1 side population (SP) cells. (A) The cells were stained with DCV dye blue-red profile. As a control, verapamil (50 μM) was added to the cells. SP cells were gated as the population of low DCV dye containing cells that disappeared upon verapamil treatment. A typical flow cytometry plot is shown where the SP fraction corresponds to 7% H1650 and 15% H1650-ER1 cells. Three independent experiments were carried out (B) H1650-ER1 SP and non SP cells were sorted and cultured for 10 days, stained with DCV dye and reanalyzed. SP cells generated SP and non SP fractions identical to H1650-ER1 cells while non SP cells generated mainly non SP cells. A representative plot is shown here. (C) Generation of tumor spheroids by SP and non SP cells. 6000 cells were seeded per well in 6 well plates and tumor spheroid generation was assessed by counting after 15 days. The error bars represent S.D. (n = 6).