Bioscope

Cells were cultured on glass-bottomed 35-mm Petri dishes (MatTek) and placed on the stage of an inverted optical microscope (Olympus IX70-S8F2) equipped with a microinbucator (HCMIS ALA Science) to hold the temperature at 37°C. A commercial stand-alone AFM (Bioscope, Veeco) adapted to the optical microscope and provided with a low-spring constant (0.03 nN/nm) AFM cantilever (Veeco) was used to assess the Young’s modulus (E) of single cells, following a protocol described in detail elsewhere (Alcaraz et al., 2003 (link), 2011 (link)). The spring constant of the cantilever was calibrated using the thermal fluctuations method (Roca-Cusachs et al., 2008 (link)). In brief, three standard force versus displacement curves (F vs. z) were recorded on the perinuclear region of each cell at moderate loading force (∼1 nN) and low speed (∼5 µm/s). The E of a single cell was computed by fitting a suitable contact elastic model to each F-versus-z curve and averaging it over the three recordings. The final E for a cell population in each experimental condition was calculated from at least nine measurements for each independent experiment (n ≥ 2).

Mechanically induced Ca²⁺ influx of individual chondrocytes was measured using a custom-built AFM/Ca²⁺ setup consisting of the atomic force microscope (Bioscope; Veeco or MFP-3D; Asylum) and a ratiometric Ca²⁺ imaging microscope (Intracellular Imaging; Fig. 2C) as described in ref. 19 (link). Briefly, AFM probe (flat, tipless) compresses single cells cyclically with trigger force of 300∼500 nN every 10 s for 2 min, and the cytosolic [Ca²⁺] transients in response to cyclical compression mechanics were measured during the loading (1 ∼2 frames). Spring constants of the tipless cantilever were 0.5 to 14 N/m (Novasan or Bruker Probes), the compression rate was 1 to 2 μm/s, and the experiments were conducted at 37 °C. The five maximum Ca²⁺ influx of each chondrocyte were determined ([Ca²⁺]_max), and influx was determined as ∆[Ca²⁺] = [Ca²⁺]_max − [Ca²⁺]_o.