Anti cd45 pe

The CTC droplet slides were blocked with PBS containing 2% BSA for 30 min, and then anti Vimentin-AlexaFluor 647 (1:200) (Cell Signaling Technology, CAT: 98565), anti-CK8/18/19-FITC (1:100) (Miltenyi Biotec, CAT: 130-080-101) and anti-CD45- PE (1:200) (Miltenyi Biotec, CAT: 130-045-801) diluted in 2% BSA were added and incubated at room temperature for 1 h. Then, the slides were washed three times with PBS, 3 min/time to remove unbound antibody. The CTC slides were mounted with the nuclear dye 4′,6-diamidino-2-phenylindole (DAPI) (Sigma–Aldrich, CAT: D9564) and observed under a fluorescence microscope (Nikon, Japan). The epithelial CTCs (E-CTCs) were defined as PanCK (+), vimentin (−), CD45 (−) and DAPI (+) cells, the mesenchymal CTCs (M-CTCs) were defined as PanCK (−), vimentin (+), CD45 (−) and DAPI (+) cells, the epithelial–mesenchymal CTCs (EM-CTCs) were defined as PanCK (+), vimentin(+), CD45 (−) and DAPI (+) cells, whereas white blood cells (WBCs) were defined as PanCK (−), vimentin (−), CD45 (+) and DAPI (+) cells, respectively. Total CTC number were calculated as the sum of E-CTCs, M-CTCs and EM-CTCs of each patient. For some patients whose blood collection does not reach 7.5 ml, the final number of CTC is calculated by the following formula: number of CTC/volume of blood used in the experiment * 7.5 ml.

Before being transferred to the well chip for analysis and retrieval, the cells were stained while they were on the surface of the porous chip. The cells were initially stained with a mixture of anti-EpCAM-FITC (Miltenyi Biotech, 130-113-263), anti-HLAG-FITC (Miltenyi Biotech, 130-111-850), and anti-CD45-PE (Miltenyi Biotech, 130-113-118) antibodies for 15 min followed by a 4 ml PBS wash. Then, the cells were also stained with a Hoechst Solution (Thermo Fisher Scientific, 62249) for 10 mins followed by another 4 min PBS wash. This was followed by the transferring of the cells to the well chip. We identified JEG3 cells as EpCAM and/or HLAG + as well as Hoechst + (Fig 3A–3D). We identified WBCs as CD45 and Hoechst + . We adopted this protocol to retrieve live cells without fixation and permeabilization, a process often used to label cytokeratin [26 (link),21 (link),22 (link),32 (link)].