Cell isolation with magnetic dynabeads

Coupling the cmHsp70.1 mAb or EpCAM mAb (CD326, clone HE125) to magnetic Dynabeads™ (Thermo Fisher, Waltham, USA) was carried out according to the manufacturer's protocol. Dynabeads coupled with cmHsp70.1 mAb were washed and incubated either with tumor cells, a mixture of tumor cells and lymphocytes or EDTA blood (7.5 ml) of patients with squamous cell carcinoma of the head and neck (SCCHN) and non-small cell lung carcinoma (NSCLC) after removal of red blood cells for 10 min at 4°C under rotation. The tube was placed in a DynaMag™ magnet and beads were separated from the supernatant after a washing step. The isolated CTCs were counted and stained with DAPI (4′,6-diamidino-2-phenylindole), cytokeratin-Alexa 488, EpCAM-PE and cmHsp70.1-FITC mAb. Isolated CTCs from tumor patients were clonally seeded in 96-well round-bottom culture plates.