Vybrant dil

Hep G2 cells and RFP-HNDFCs were mixed at 1.0 × 10⁵ cells per cell type in 2 mL of culture medium. Aptamer-conjugated oligopeptide SAMs were prepared using mixtures of peptide-N₃ and peptide-COOH at 0.1%, 1%, and 10% peptide-N₃. The cell mixture was poured on the aptamer-conjugated SAMs. After 3 h of culture, non-attached cells were washed twice with PBS and the number of each type of cells was counted on microscopic images using ImageJ software. SAMs without conjugation of aptamers and bare gold surfaces were used as controls. To obtain a separation curve through multistep enrichment of target cells, separation with the oligopeptide SAM was examined using cell suspensions at various ratios of Hep G2 cells to RFP-HNDFCs or normal human hepatocytes. In both cases, the cell density was 2.0 × 10⁵ cells/2 mL/well. To distinguish Hep G2 cells from normal human hepatocytes, Hep G2 cells were previously stained with Vybrant-Dil (Thermo Fisher, Japan) in DMEM without serum for 15 min at 37 °C, followed by washing thrice with DMEM supplemented with 10% FBS. The number of fluorescently labeled and non-labeled cells was counted on microscopic images.

The melanoma cells were stained with Vybrant DiL (Thermo Scientific, Dreieich, Germany) for 15 min at 37 °C before spheroid formation was initiated as described above. Plasma treatment was performed on day four, followed by incubation for 4 h. Spheroids (with residual amounts of original medium) were transferred to a fresh round-bottom multiwell plate without adding new culture medium and overlaid with 50 µL of matrigel (Corning, Wiesbaden, Germany). Plates were centrifuged at 500× g for 5 min in a precooled centrifuge (4 °C). matrigel was allowed to polymerize for 1 h at 37 °C, and 100 µL of culture medium was added. Spheroids and outgrowths of single melanoma cells were visualized in real-time using high-content imaging for 72 h. Fresh culture medium (50 µL) per well was added on day 2.