Epics altra flow cytosorter

Forty-eight hours after transfection with specific siRNAs, CNE2 and 5-8F cells were trypsinized and resuspended at density of 1 × 10⁶ cells per ml. The DNA-binding dye, Hoechst 33342 (Sigma-Aldrich), was then added at a final concentration of 7.5 μg/ml and incubated for 90 min in the dark with periodic mixing. After washing twice with PBS, the cells were placed at 4 °C in the dark before flow cytometry (EPICS ALTRA Flow Cytosorter, Beckman Coulter, Indianapolis, IN, USA) using dual-wavelength analysis. However, a fraction of the cell preparation was incubated with 5 μM Ko143 (Ko143, a specific inhibitor of ABCG2, Sigma-Aldrich) for 10 min at 37 °C before adding Hoechst 33342 to determine whether Ko143 would block the fluorescent efflux of SP cell.

48 hours after transfection with specific siRNAs, 6-10B, and SUNE1 cells were trypsinized and resuspended at a density of 1 × 10⁶ cells/ml. The DNA binding dye, Hoechst 33342 (Sigma-Aldrich), was then added at a final concentration of 5 μg/ml and incubated for 90 min in the dark with every half an hour mixing. Then the cells were washed with PBS for twice. The cells were placed at 4°C in the dark before flow cytometry (EPICS ALTRA Flow Cytosorter, Beckman Coulter) using dual wavelength analysis. While, a portion of the cells was incubated with100ug/ml verapamil (a calcium ion tunnel antagonist, Sigma-Aldrich) for 30 min at 37°C prior to adding Hoechst 33342 to determine whether this would block the fluorescent efflux of SP cells in the 6-10B and SUNE1 populations.

The CNE-2-S-18 and CNE-1 cells or single cells obtained from primary xenograft tumors were harvested and resuspended at a concentration of 1 × 10⁶ cells/ml. Hoechst 33342 was then added to a final concentration of 5 μg/ml and incubated for 90 min at 37°C in the dark with interval mixing. After washing twice with PBS, the cells were kept at 4°C in the dark before flow cytometry analysis (EPICS ALTRA Flow Cytosorter, Beckman Coulter). Meanwhile, a subset of the cells was incubated with 5 μM FTC for 5 min at 37°C prior to adding Hoechst 33342. Flow cytometry data were analyzed using FlowJo software.