Expression of FACS-sorted CD133+ and CD133- cells of three gastric cell lines

To identify the gene expression signature associated with CD133, the well-known stem cell markers, three gastric cancer cell lines were obtained (KATO-III, SNU201 and SNU601). Cultured gastric cancer celllines were sorted into CD133+ and CD133- population by FACS sorting and microarray-based gene expression profiling was performed. Three GC celllines (KATO-III, SNU201 and SNU601) were purchased from the Korean Cell Line Bank and maintained in RPMI1640 medium supplemented with 10% calf serum. Cells were harvested and incubated in cell-staining buffer containing phycoerythrin (PE)-labeled anti-CD133/1(AC133) antibody. An isotype-matched PE-labeled control antibody was further employed to label the samples and set gating levels. The top 10% of CD133+ cells, in terms of fluorescence intensity, and the bottom 6% of CD133- cells, were collected via MoFlo XDP flow cytometry, which was also employed to sort cell lines into CD133+ and CD133- populations. Total RNA was isolated from sorted cells. The extracted RNA was further amplified and biotinylated using an Illumina TotalPrep RNA Amplification Kit. Extracted RNA was quantified on an Agilent 2100 Bioanalyzer. A Whole-Genome Expression Direct Hybridization Kit (Illumina) was used to hybridize 750 ng of cRNA from each sample to Human HT-12 v3 expression BeadChips (Illumina) at 58°C overnight. Unbound probe was removed by vigorous washing and the BeadChip was scanned with a BeadArray reader (Illumina).