Cellsearch ctc kit

A solution of H1975 cells stained with Cell Tracker™ Green (Thermo fisher scientific) was loaded on the single-cell picking system and single cells were collected and added to individual wells of a 96-well microplate. The existence of a single cell in each well was confirmed by fluorescent microscopy. To quantify tumor cells identified using the single-cell picking system, approximately 1500 or a small number of (2–25) A549 cells were spiked into 7.5 mL of whole blood from a healthy donor (HD), which was collected in a CellSave Preservative Tube (Janssen Diagnostics). A549 cells spiked in HD blood were processed using the CellSearch® CTC Kit (Janssen Diagnostics), and A549 cell counts were determined using the CellTracks Analyzer II® (Janssen Diagnostics). Enriched cells were loaded onto the single-cell picking system and re-counted. CTC counts obtained by CellSearch® and the single-cell picking system were compared. When a small number of cells, i.e., A549 cells, were spiked, single cells were recovered and the recovery rate was calculated.

The enrichment and enumeration of tumor cells from whole blood were performed using the FDA-approved CellSearch® System (Janssen Diagnostics, Raritan, NJ, USA). First, 7.5 mL of the whole blood sample was processed using the CellSearch® CTC Kit (Janssen Diagnostics). In this assay, EpCAM-based immunomagnetically enriched cells were fluorescently counterlabeled with DAPI to stain nuclei, phycoerythrin (PE)-conjugated antibodies directed against cytokeratins 8/18/19, and allophycocyanin (APC)-conjugated antibodies directed against CD45 to stain the remaining WBCs. After enrichment, isolated fluorescently labeled cells were resuspended in a MagNest^Ⓡ Cartridge Holder (Janssen Diagnostics) and analyzed (i.e., identified and enumerated) using the CellTracks Analyzer II^Ⓡ (Janssen Diagnostics) according to the manufacturer’s instructions.