Anti ck8 18 19 fitc

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.

Cells from tumour sample were divided into aliquots, single-stained with SYTOX green (Invitrogen, Carlsbad, CA, USA), anti-CD326-PE (Miltenyi Biotec, Germany), anti-Ber-EP4-FITC (Dako, Germany), anti-CK7/8-FITC (BD Biosciences CA, USA) and anti-CK8/18/19-FITC (Miltenyi Biotec, Germany) for 10 min. Stained cells were washed and resuspended in PBS. Non-stained and each single-stained sample were prepared for fluorescent compensation. For all experiments, analysis of cells used a FACSCalibur instrument (BD Bioscience, CA, USA) and FCS Express software (De Novo, Los Angeles, CA, USA).