2.2. Cell Culture and Adhesion Assay
Melanoma cell lines including M21, M21-L, and B16 were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin/streptomycin (P/S) under 5% CO2 at 37 °C. Human prostate cancer cells, LNCaP and PC3, were cultured in RPMI supplemented with 10% FBS and 100 units/mL P/S under 10% CO2 at 37 °C. All cells were plated into 100-mm culture dishes (Corning, Tewksbury, MA, USA) in 10 mL of pre-warmed culture media. When the cells reached 70%–80% confluence at a density of 2–5 × 106 cells/dish, either ESNF10 (700 nm NIR fluorophore) [24] or IR786 (800 nm NIR fluorophore) [21] was added to the dish at 2 µM in media. After 20 min incubation at 37 °C, the cells were washed twice with media and the NIR fluorescence signals were observed under a multi-channel fluorescence microscope (see below). The cells were then trypsinized and seeded onto the ligand-bearing surface in DMEM containing 10% FBS at 37 °C. Incubation parameters, including the effect of rocking (0 vs. 30 rpm), incubation time (30–180 min), presented ligand concentration at the time of spotting (0.1–1.0 mM), and applied cell density (0.2 × 106−8 × 106 cells), were systematically optimized. After incubation, the slides were gently washed with cell culture media before scoring.