Vybrant did red fluorescent dye

To enable convenient cell imaging and counting, cultured EpCAM-positive NSCLC cells (1 × 10⁶ ml⁻¹) were prestained with a Vybrant DiO green fluorescent dye (Invitrogen) in serum-free culture medium at 37°C for 1 hour. EpCAM-negative WBCs were isolated from blood samples of the healthy donors with approval from the University of California, Los Angeles Institutional Review Board (no. 00000173) and then prestained with a Vybrant DiD red fluorescent dye (Invitrogen). Excess cell-labeling dye was removed by centrifuging the labeled suspension at 1500 rpm for 5 min and washed with PBS twice. Typical artificial CTC samples were prepared by spiking 200 prestained NSCLC cells into the prestained WBCs (5 × 10⁶ cells ml⁻¹) in 200 μl of RPMI 1640. The artificial CTC sample was incubated with TCO–anti-EpCAM (0.1 ng) in RPMI 1640 (200 μl) at room temperature for 30 min and then centrifuged at 300g for 10 min to remove the excess TCO–anti-EpCAM and nonreactive TCO-PEG₄-NHS ester. The resulting samples containing TCO-grafted CTCs were then purified using Click Chips. For CTC enumeration, the cells captured on Tz-grafted SiNWS were stained with DAPI and imaged under a fluorescence microscope (Nikon 90i, W₁, 325 to 375 nm; W₂, 465 to 495 nm; W₃, 590 to 650 nm).