Journal: British Journal of Cancer

Article Title: Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting

doi: 10.1038/bjc.2013.92

Figure Lengend Snippet: Schematic of overall experimental workflow. Step 1: Tumour cells of three different human cultured breast cancer cell lines were spiked in healthy donor blood at a concentration of 10 3 tumour cells per 7.5 ml whole blood. Step 2: Tumour cells were immunomagnetically enriched using either the CellSearch CTC kit or the CellSearch Profile kit followed by a manual staining procedure. Step 3: Cells were reconstituted in a final volume of 14 μ l and loaded in a DEPArray cartridge. Step 4: Analysis and sorting procedures were performed on the DEPArray system. Step 5: Single cells and groups of cells of interest were isolated with the DEPArray system. Step 6: Mutation or transcriptional analysis of isolated tumour cells.

Article Snippet: The sample was processed with the CellSearch CTC kit and the CellSearch cartridge was stored at 4 °C for 8 days.

Techniques: Cell Culture, Concentration Assay, Staining, Isolation, Mutagenesis

Journal: British Journal of Cancer

Article Title: Semiautomated isolation and molecular characterisation of single or highly purified tumour cells from CellSearch enriched blood samples using dielectrophoretic cell sorting

doi: 10.1038/bjc.2013.92

Figure Lengend Snippet: Composite image gallery showing staining characteristics of MDA-MB-231 cells and WBCs from a CellSearch cartridge after immunomagnetic enrichment with the CellSearch CTC kit visualised on the DEPArray system. ( A ) Tumour cells were defined using standard CellSearch CTC criteria being (1) round to oval shape, (2) presence of a clear DAPI-stained nucleus, (3) at least 50% overlap between the CK-PE-positive cytoplasm and the nucleus and (4) CD45-APC negativity. Separate images for PE, DAPI and APC fluorescence and bright field channels and merged CK-PE/DAPI and CD45-APC/DAPI images of three single MDA-MB-231 cells (top 3 rows) and two WBC (bottom 2 rows) are shown. ( B ) Scatter plot of mean fluorescence intensities for CK-PE (y axis) and CD45-APC (x axis) staining by the DEPArray software. Rectangular gates can be applied to aid quick identification of cells or populations of cells for cell sorting. Green dots represent CK+/CD45− MDA-MB-231 cells selected by the user. Red dots represent CK−/CD45+ WBCs. Grey dots represent unselected events.

Article Snippet: The sample was processed with the CellSearch CTC kit and the CellSearch cartridge was stored at 4 °C for 8 days.

Techniques: Staining, Fluorescence, Software, FACS

Journal: Cancers

Article Title: Assessment of a Size-Based Method for Enriching Circulating Tumour Cells in Colorectal Cancer

doi: 10.3390/cancers14143446

Figure Lengend Snippet: Qualitative assessment of MetaCell’s ability to retain CRC cells spiked into blood. After filtration, the cells retained on the MetaCell membrane from the following samples were stained with Hoechst 33,342 (nuclear stain), white blood cell (WBC) markers (CD45 and CD16), and CTC markers (EpCAM and cytokeratins): unspiked healthy blood (negative control, top row), healthy blood spiked with CRC cells (middle row), and unfiltered CRC cells (positive control, bottom row). The representative images show that unspiked healthy blood cells retained after filtering were positive for WBC markers and lacking CTC markers. Both healthy blood spiked with CRC cells retained on the MetaCell membrane and unfiltered CRC cells were positive for Hoechst 33,342 and CTC markers but negative for WBC markers. Cells were visualised on a Nikon A1+ confocal microscope at 200× magnification. Scale bar represents 80 µm.

Article Snippet: The MetaCell CTC kit is a size-based CTC enrichment method which has previously been used to enrich and culture CTCs in vitro from a multitude of cancer types, including breast, gynaecological, lung, esophageal, gastric, and urothelial cancers [ , , , , , ].

Techniques: Filtration, Staining, Negative Control, Positive Control, Microscopy

Journal: Cancers

Article Title: Assessment of a Size-Based Method for Enriching Circulating Tumour Cells in Colorectal Cancer

doi: 10.3390/cancers14143446

Figure Lengend Snippet: Quantitative analysis of CTC markers in unfiltered and MetaCell-filtered CRC cells. ( A ) Expression of CTC markers ( EPCAM and KRT20 ) for unfiltered (HCT116_UF) and MetaCell-filtered HCT116 cells (HCT116_MF). ( B ) Expression of CTC markers for unfiltered DLD1 cells (DLD1_UF) and MetaCell-filtered DLD1 cells (DLD1_MF). Error bars represent the mean ± SD of three biological replicates. p -values determined by paired Student’s t -test; ns p > 0.05.

Article Snippet: The MetaCell CTC kit is a size-based CTC enrichment method which has previously been used to enrich and culture CTCs in vitro from a multitude of cancer types, including breast, gynaecological, lung, esophageal, gastric, and urothelial cancers [ , , , , , ].

Techniques: Expressing

Journal: Cancers

Article Title: Assessment of a Size-Based Method for Enriching Circulating Tumour Cells in Colorectal Cancer

doi: 10.3390/cancers14143446

Figure Lengend Snippet: MetaCell recovery rates for healthy blood spiked with different concentrations of CRC cells. Different numbers (10, 100, and 500) of either HCT116 cells ( A ) or DLD1 cells ( B ) were spiked into healthy blood samples and filtered with MetaCell. The cells retained on the membrane and the same number of unfiltered cells were immunostained with CTC (EpCAM or cytokeratins) and WBC markers (CD45 and CD16). Cells that stained positively for Hoechst 33,342 and and at least one of the CTC markers but did not stain for WBC markers were manually counted using a Nikon A1+ confocal microscope at 200× magnification. Recovery rates are represented as the mean ± SD of three biological replicates. p -values were determined by a paired t -test. ns p > 0.05.

Article Snippet: The MetaCell CTC kit is a size-based CTC enrichment method which has previously been used to enrich and culture CTCs in vitro from a multitude of cancer types, including breast, gynaecological, lung, esophageal, gastric, and urothelial cancers [ , , , , , ].

Techniques: Staining, Microscopy

Journal: Cancers

Article Title: Assessment of a Size-Based Method for Enriching Circulating Tumour Cells in Colorectal Cancer

doi: 10.3390/cancers14143446

Figure Lengend Snippet: Gene expression analysis to evaluate the effect of cell numbers on the recovery rates for MetaCell. The gene expression analysis of CTC markers ( EpCAM and KRT20 ) for the different numbers of unfiltered (10, 100, and 500 UF) and MetaCell-filtered HCT116 ( A ) or DLD1 ( B ) cells (10, 100, and 500 MF). Error bars represent the mean ± SD of three biological replicates. p -values were determined by paired t -tests. ns p > 0.05 for both cell lines.

Article Snippet: The MetaCell CTC kit is a size-based CTC enrichment method which has previously been used to enrich and culture CTCs in vitro from a multitude of cancer types, including breast, gynaecological, lung, esophageal, gastric, and urothelial cancers [ , , , , , ].

Techniques: Expressing