C10600

Cells were loaded with 5 μM Fura-2/AM in DMEM at 37 °C for 30 min. Ratiometric Ca²⁺ imaging was performed at 340 and 380 nm in 2 mM Ca²⁺ Tyrode’s solution (129 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 30 mM glucose, and 25 mM Hepes, pH 7.4) or cultued medium with a IDX81 microscope (Olympus) equipped with an Olympus x40 oil (NA 1.30) objective equipped with a fluorescent arc lamp (LAMDA LS), excitation filter wheel (SUTTER, LAMBDA 10–2), stage controller (ASI, MS-2000) and a CCD camera (HAMAMATSU, C10600) at room temperature. Images were processed with Metamorph and analyzed with Igor Pro.

Cell probes were prepared using triangular shaped tipless cantilevers (microlevers) (catalog no. NP-O10; Bruker Corporation) coated with bioinspired polydopamine wet adhesives. Cantilevers were immersed for 1 h in a 10 mM Tris buffer solution (pH 8.5) containing 4 mg ml⁻¹ dopamine hydrochloride (99%; Sigma) and dried with N₂ flow. Single cells were then attached onto the polydopamine-coated cantilevers using a Bioscope Catalyst AFM (Bruker Corporation). Hydrophobic substrates were prepared by immersing gold-coated substrates overnight in solutions of 1 mM 1-dodecanethiol (Sigma-Aldrich) (98%), rinsing them with ethanol, and drying them under N₂. To have cell aggregates on the hydrophobic surface, we deposited a drop of a cell suspension and allowed it to sediment for 10 to 15 min, and the cells were covered with 4 ml of PBS. Then, the cantilever was brought into contact with an isolated cell for 3 min, and the obtained cell probe was then transferred over a cell aggregate for cell-cell force measurements. The nominal spring constant of the cantilever was ∼0.06 N m⁻¹ as determined by the thermal noise method. Single-cell force spectroscopy measurements were performed at room temperature (20°C) in PBS, using a Bioscope Catalyst combined with an inverted optical microscope (Zeiss Axio Observer Z1 equipped with a Hamamatsu camera C10600 [Oberkochen, Germany]).