Vysis centromere probe cep8 spectrum orange

Experiment was performed according to the product manufacturer’s instruction (Cytelligen, San Diego, CA, USA) (31 (link)). Briefly, samples on the coated slides were subjected to Vysis Centromere Probe (CEP8) Spectrum Orange (Abbott Laboratories, Abbott Park, IL, USA) hybridization for 3 hours using the S500 StatSpin ThermoBrite Slide Hybridization/Denaturation System (Abbott Molecular, Des Plaines, IL, USA). Samples were subsequently incubated with Alexa Fluor 594–conjugated monoclonal anti-CD45 and Alexa Fluor 488–conjugated monoclonal anti-EpCAM antibodies (Cytelligen, San Diego, CA, USA). Cell nuclei were stained with DAPI (Life Technologies, Carlsbad, CA, USA). Images of the identified tumor cells were automatically acquired using a Metafer-iFISH automated Circulating rare cells (CRCs) image scanning and analysis system (31 (link)). CTCs and DTCs were defined as DAPI⁺, CD45⁻, heteroploid chromosome 8 cells with or without visible EpCAM or diploid CEP8 signal with visible EpCAM. Cell clusters were defined as ≥2 contacted cells. To avoid bias, blood sample collection and CTC and DTC detection were coperformed by cross-blinded physicians and research scientists.