An example of cell sorting.

<p>Two photographs of the discarded reservoir (a) and the collection reservoir (b) indicated in the chip photograph are shown. Clustered cells are indicated by white arrows. Bars, 100 µm.</p

Results of quantitative gene copy number assays.

<p>(a) Results of CGH assays performed for the MAT-LyLu cell line. Liver tissue of the rat was used as a reference. Gene amplifications for <i>csrp2</i> and <i>zdhhc17</i> located on chromosome 7q13 were found. (b) Results of TaqMan copy number assays performed with clusters larger than 300 µm² collected in the collection reservoir, and cells smaller than 300 µm² collected in the discarded reservoir. Results of the assays for the MAT-LyLu cell line (positive control) and liver tissue (negative control) are also shown.</p

Summary of image processing.

<p>Firstly, photographs of both a cell and the background were taken. Next, the background image was subtracted from the cell image and holes were filled. Finally, imaging biomarkers, <i>S</i>, <i>S_n</i>, <i>N_n</i>, and <i>R</i>, were calculated. Bars, 10 µm. The hole-filling procedure is explained as indicated by an asterisk. Bar, 10 µm.</p

Typical cell images for clustered cells composed of more than three cells in cancer cell-implanted and control blood.

<p>Each data count (<i>n</i>) indicates the image number having the same cluster size and <i>N_n</i>. Bars, 20 µm.</p

Typical cell images for single and double cells in cancer cell-implanted and control blood.

<p>Each data count (<i>n</i>) indicates the image number having the same cluster size and <i>N_n</i>. Bars, 20 µm.</p

Instrumental set-up.

<p>(a) Summary of the on-chip multi-imaging flow cytometry system. The system was composed of seven major modules: (i) microchip, (ii) bright-field (BF) imaging, (iii) fluorescent (FL) detection, (iv) multi-view, (v) CCD camera, (vi) sorting, and (vii) controller, as numbered in the figure. (b) Summary of the multi-view module. (c) A photograph of the system.</p

Summary of total cell area, <i>S</i>, total nucleus area, <i>S_n</i>, number of nuclei, <i>N_n</i>, and perimeter ratio, <i>R</i>, for each cluster size.

<p>Summary of total cell area, <i>S</i>, total nucleus area, <i>S_n</i>, number of nuclei, <i>N_n</i>, and perimeter ratio, <i>R</i>, for each cluster size.</p

Summary of the number of nuclei, <i>N_n</i>.

<p>(a) A histogram of <i>N_n</i> obtained from cancer cell-implanted and control blood. (b) The relationship between <i>N_n</i> and cell cluster size.</p

Histograms of total cell area, S, for cancer cell-implanted (a and c) and control blood (b and d).

<p>Two threshold values (a) and (b) for cluster identifications are indicated as dotted and dashed lines.</p

Comparison of protein and mRNA expression levels of various factors between low and highly metastatic gastric cancer cell lines.

<p>(A) Western blot analysis of total cell lysates shows protein expression levels of NDRG1, growth factor receptor, EMT-related proteins, Wnt/β-catenin-related proteins, and other factors in HSC-58, 58As1 and 58As9 cells. (B) Comparison of mRNA expression levels of NDRG1, E-cadherin, vimentin, Snail, MMP-1 and β-catenin in HSC-58, 58As1 and 58As9 cells by qRT-PCR analysis. (C) Immunocytochemical analysis of E-cadherin and β-catenin in HSC-58 and 58As9, using specific antibodies against E-cadherin, β-catenin and DAP1. Magnification×200. (D) Western blot analysis shows expression of β-catenin and Snail in nucleus and cytosol fraction. CREB, a nuclear marker, and α-tubulin, a cytosol marker. (E,F) Comparison of luciferase activity driven by E-cadhrin promoter and β-catenin (TopFlash) driven promoter between HSC-58 and its highly metastatic cell lines. The relative promoter activity is presented when normalized by the activity in HSC-58. *p<0.01.</p