Additional file 1.

Figure S1 Fluorescence activated cell sorting (FACS) analysis of two representative clinical blood samples positive for CTCs. Samples after immunomagnetic enrichment (enriched for tumor cells) were subjected to fluorescence activated cells sorting (FACS) to isolate for CTCs. Tumor cells were stained with EpCAM (EBA-1) mAb conjugated to phycoerythrin (EpCAM-PE), a nucleic acid dye and a leukocyte-specific CD45 (2D1) mAb conjugated to peridinin-chlorophyll-protein-Cy5.5 (CD45-PerCPCy5.5). Forward and side scatters (top left panel) were used for preliminary identification of cells (P1 gate) and to exclude debris. P2 gate (top right panel) was used to select for nucleated (a nucleic acid dye+) cells while gates in the lower panels were used to select for EpCAM + and CD45- (P3 gate) and EpCAM + and nucleated (a nucleic acid dye+) cells (P4 gate). CTCs must be present within gates P1 to P4, and were defined as EpCAM+, CD45-, and nucleated (a nucleic acid dye+). P5 gate (lower left panel) was used to select for CD45+, EpCAM-, and nucleated (a nucleic acid dye+) cells when sorting for leukocytes. Forward scatter (FSC) vs side scatter (SSC) plots are on a linear scale while SSC vs nucleic acid dye plots are on a semi-logarithmic scale. EpCAM-PE vs CD45-PerCP-Cy5.5 and EpCAM-PE vs nucleic acid dye are log-log plots.

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Magbanua et al. BMC Cancer 2012 12:78   doi:10.1186/1471-2407-12-78