CTC Enrichment and Enumeration Protocol

Samples are filtered with a CellSieve™ Microfiltration Assay using a low-pressure vacuum system which isolates CTCs based on size exclusion, >7 micron, as previously described1 (link)2 (link)3 (link)25 (link)39 . Cells were stained and identified by fluorescent enumeration using CTC enumeration stains, as previously described. Briefly, a low-pressure system uses a filter holder assembly with CellSieve™ filter attached. Peripheral blood (7.5 mL), is diluted in a prefixation buffer and drawn through the filter. The filter is washed, postfixed and permeabilized. The captured cells are stained with an antibody cocktail consisting of FITC-anti-Cytokeratin 8, 18, 19, PE-anti-EpCAM and Cy5-anti-CD45 for 1 hour and mounted with Fluoromount-G/DAPI (Southern Biotech). An Olympus BX54WI Fluorescent microscope with Carl Zeiss AxioCam was used to image the samples. Exposures were preset as 2 sec (Cyanine5), 2 sec (PE), 100–750 msec (FITC), and 10–50 msec (DAPI) for equal signal comparisons between cells. A Zen2011 Blue (Carl Zeiss) with AxioVision Mark and Find module was used to process the images, mark the x/y placement of the cells, and relocate previously imaged cells in a semi-automated manner. Samples were archived and placed in storage at 4 °C for 1 week-2 years.