8 well micro slide

Cells were seeded either into 8 well microslides (Ibidi) with 3 × 10⁴ cells per well in 150 μL of growth medium for live-cell microscopy or on 8 spot slides (Thermo Scientific) with 8 × 10⁴ cells/mL for fixed-cell experiments and left to recover for 24 h at 37 °C and 5% CO₂. For the experiment, cells were incubated first with serum-free media for 2 h at 37 °C and then, after washing, treated with Cy5–Y-1 (0.5 μM) for living cells and biotin–Y-1 or biotin–Y-sc1 (1 μM) for fixed-cell experiments in 1% BSA in PBS for 10 min at 37 °C. After a short wash with serum-free phenol-free media, cells stained with Cy5–Y-1 were imaged quickly at the confocal Zeiss LSM 700 Olympus microscope (Carl Zeiss AG, Oberkochen, Germany). Cells incubated with biotin–Y-1 were, after washing with PBS, fixed with 4% paraformaldehyde for 20 min on ice, and permeabilized with 0.5% Triton X-100 for 15 min on ice. After another washing step, cells were incubated with 1:200 FITC–avidin (no. 434411, Life Technologies) in 20% FCS in PBS for 20 min on ice and washed again. DAPI (1 μg/mL) in PBS was used as a counterstain with Vectashield (H-1000, Vector Laboratories, Inc., Burlingame, CA) for mounting. Fixed and stained cells were then imaged at the confocal Zeiss LSM 700 Olympus microscope (Carl Zeiss AG, Oberkochen, Germany), and the fluorescence intensity of biotin–Y-1 per cell area was evaluated with ImageJ.