CTC Isolation and Identification Protocol

For human samples, 7.5 mL of whole blood was processed on the CellSearch® Autoprep system using the CellSearch® CTC kit (Menarini Silicon Biosystems), analyzed on the CellSearch® Analyzer, and assessed for the presence of CTCs as previously described [11 (link)]. For mouse samples, 50 µL of whole blood was incubated with components of the CellSearch® CTC kit including anti-EpCAM ferrofluid (25 µL), Capture Enhancement Reagent (25 µL), Nucleic Acid Dye (50 µL), Staining Reagent (50 µL), Permeabilization Reagent (100 µL), as well as anti-mouse CD45-APC (1.5 µL) (eBiosciences, San Diego, CA) as described previously [11 (link)]. Samples were manually immuno-magnetically separated and transferred to a CellSearch® MagNest™ cartridge for analysis using the CellSearch® Analyzer. In all cases, cells displaying the phenotype of EpCAM⁺/CK⁺/DAPI⁺/CD45⁻ cells with a round/oval morphology were classified as CTCs.