Cellsearch ctc kit

Circulating tumor cells were detected through two methods, one EpCAM-dependent (CellSearch) and the other size based (ScreenCell). Peripheral blood samples were drawn after informed consent into two tubes, after discarding the first milliliters to avoid contamination with cutaneous epithelial cells taken by the needle during the sampling. 7.5 mL of blood was collected in CellSave preservative tube (Menarini Silicon Biosystems, Castel Maggiore, Bo, Italy) and processed by CellSearch (Menarini Silicon Biosystems), employing CellSearch CTC kit, according to manufacturer's instructions. Six milliliters of blood was collected in tube containing K₂EDTA and processed by ScreenCell Cyto kit (ScreenCell, Sarcelles, France) to isolate fixed cells for cytological studies. Briefly, in order to fix the cells and lyse red blood cells, 3 mL of blood was diluted in 4 mL of FC2 filtration buffer (ScreenCell). After 8 min of incubation at room temperature, 7 mL of diluted sample was added into device tank and filtered under a pressure gradient using a vacutainer tube. After washing with PBS to remove red blood cells debris, the filter was left on absorbing paper to dry at room temperature and then stored at −20°C. The filtration was carried out in duplicate and completed within 3 min.

Aliquots of 7.5 mL of blood were processed with the CellSearch^® system (Menarini Silicon Biosystems, Castel Maggiore (BO), Italy) for CTC detection. CellSearch analysis was performed within 96 h after blood was drawn. Antibodies directed against the epithelial cell adhesion antigen (EpCAM) coupled to ferrofluids were used to enrich CTC from the background of leukocytes. The enriched cells were fluorescently labeled with the CellSearch CTC kit (Menarini Silicon Biosystems) using the nucleic acid dye 4′6-diaminodino-2-phenylindole (DAPI) for DNA staining, anti-cytokeratin monoclonal antibodies (mAbs) C11 and A53.B/A2 labelled with phycoerythrin (PE), and anti-CD45 mAb (clone HI30) labeled with allophycocyanin (APC) for recognizing leukocytes. Peridinin Chlorofyll A Protein (PerCP) labeled mAb anti-CD16 (clone 3G8, Biolegend, San Diego, CA, USA) directed against granulocytes, and Alexa-Fluor488 conjugated to the lectin wga (Thermo Fisher Scientific, Waltham, MA, USA) were added to the extra marker position in the CellSearch Epithelial Cell kit with an end concentration of 2 µg/mL and 3 µg/mL, respectively. This extra marker channel was used for the measurement of PerCP and DIOC on the CellTracks Analyzer II, which generates images of the complete cartridge for all channels.

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.