2.3 84-1+ cell selection

The selection and validation methods used to isolate and enrich 84-1+ cells were previously described [15]. Briefly, an antibody against human Fc receptor (Miltenyi Biotec, Auburn, CA) was added to the PBMCs cell suspension to reduce nonspecific binding. CD45+ immune cells were then depleted using an EasySep human CD45 depletion kit (StemCell^™ Technologies) according to the manufacturer’s recommendation. Later, the CD45- cell fractions from the blood were labeled with the 84-1 anti-vimentin antibody, followed by the addition of mouse IgG-binding microbeads (Miltenyi Biotec) to the mixture after incubation. The labeled cells were then extracted using the magnetic column according to the manufacturer’s protocol (Miltenyi Biotec). The 84-1+ and CD45- cells obtained were CSV+ CTCs ready for further analysis (Fig. 1D).