Cytokeratin fitc

Calcein AM (BIOTIUM, USA) was used to stain all the MCF-7 cells, and Hoechst (Life, USA) was used to stain the DNA in all the cell nuclei. Labeling was completed by two approaches: one, by putting the staining reagents into the assay directly, and the other by putting into the cell suspension. Next, 1 mL of 1× PBS containing 10 mg mL⁻¹ (1%) bovine serum albumin (BSA, Solarbio, China) and 0.05% Tween-20 was used with cancer cells to reduce the non-specific cell adhesion on the surface of the structure. For cells captured on the Hoechst chip, Cytokeratin-FITC (BD Biosciences) and CD45-PE (BD Biosciences) were used for on-chip staining. A small limited number of unlabeled tumor cells were spiked into the lysed and whole blood. Those artificial patient bloods were processed through the chip followed by washing with PBS, fixing, and permeabilization. Anti-cytokeratin (BD Biosciences) and anti-CD45 (BD Biosciences) and Hoechst applied in 1% bovine serum albumin were utilized for all the samples. After washing, then the samples were ready for microscopic imaging.^{45 (link)}

MNCs were stained with the following antihuman antibodies: Propidium Iodide Staining Solution (BD pharmingen, Catalog no. 556473), CD45‐BV421 (BD Horizon, Catalog no. 563879), CEA‐FITC (BIO‐RAD, Catalog no. MCA1744F), EpCAM‐APC (BD Biosciences, Catalog no. 347200), and Cytokeratin‐FITC (BD Biosciences, Catalog no. 347653). Labeled cells were analyzed using FACSAria^TM (BD). Dead cells were excluded by PI staining, and then, CD45 negative cells were analyzed by positivity of CEA, CK, and EpCAM. Isotype IgG antibody for anti‐CEA, anti‐EpCAM, and anti‐CK antibodies were used for control, and set the cutoff value for the positivity. We divided samples for each following staining; (a) CK and EpCAM, (b) CEA and EpCAM, and (c) isotype IgG. The number of cells for each fraction was recalculated to get the number of cells per 10 ml blood.