Live Cell Staining and Sorting Protocol

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)].