4.2. Fluorescent Probe Labeling to EGFR

Anti-EGFR antibody-conjugated fluorescent nanoparticles were used to label EGFR for tracking. The FN-IgG probe was prepared as described previously [12,13]. Biotinylated monoclonal anti-EGFR antibodies (MS-311-B, Thermo Fisher Scientific, Waltham, MA, USA) were mixed at 1:1 ratio with 40 nm NeutrAvidin-labeled red fluorescent nanoparticles (F8770, Thermo Fisher Scientific, Waltham, MA, USA) in 1.5% BSA/PBS solution (BSA, S7806, Sigma-Aldrich, St. Louis, MO, USA), respectively. The antibody-conjugated fluorescence nanoparticles (~30 nM, the stock solution) can be stored at 4 °C for up to 1 week. The number of antibodies per nanoparticle should follow a Poisson distribution. The brightness of single fluorescent nanoparticles was characterized using both fluorescence correlation spectroscopy and the TSUNAMI microscope. The photon count rates of the 40 nm red fluorescent beads were in the range of 200–500 kHz. For EGFR tracking, cells were seeded onto optical imaging 8-well chambered coverglasses (155409, Thermo Fisher Scientific, Waltham, MA, USA) with a cell density of 1 × 10⁵ cells per well and allowed to grow to ~70% cell confluency. Before tracking experiments, cells were stained with a mixture of Hoechst 33258 (H3569, Thermo Fisher Scientific, 1:1000 dilution in DMEM) and CellMask™ Deep Red ({"type":"entrez-nucleotide","attrs":{"text":"C10046","term_id":"1535117","term_text":"C10046"}}C10046, Thermo Fisher Scientific, 1:1000 dilution in complete medium) for 10 min at 37 °C. After membrane staining, the staining buffer was replaced with the EGFR-labeling solution (antibody-conjugated fluorescent nanoparticles at 100 pM) diluted from the stock solution (30 nM). The reaction was incubated for 30 min at 37 °C. The EGFR-labeling solution was removed, and the samples were washed twice using PBS to remove the unbound fluorescent nanoparticles. Upon completion of membrane staining and EGFR labeling, the chambered coverglass was immediately put on the TSUNAMI microscope for tracking experiments. Two to four trajectories (duration ranged from 1–10 min) were typically obtained from each well. The volumes of all solutions and washing buffers used in staining were 200 µL per well.