Fura 2

Cells were loaded with 10 μM Fura2-AM (Dojindo, Kumamoto, Japan) in SBS for 30 min at 24–26 °C, and thereafter, superfused with SBS for 10 min to washout the Fura-2AM. Fura-2 fluorescence signals were measured every 5 s using the Argus/HisCa imaging system (Hamamatsu Photonics, Hamamatsu, Japan), driven by Imagework Bench 6.0 (INDEC Medical Systems, Santa Clara, CA, USA). For each analysis, the whole cell area was chosen as the region of interest to average the fluorescence ratio. For quantitative measurement of change in Ca²⁺ response, we collected 50 and 20 single cells on one coverslip for analysis of HEK and MC3T3-E1 cells, respectively, and repeated the same experiment with the other coverslips to reduce variation. For constructing a concentration-response curve, a set of the summarized data was fitted to a standard Hill equation (Origin J9.1, LightStone, Tokyo, Japan). To apply laminar fluid flow, cells were maneuvered to the exit of a thin capillary tube with tip diameter of 350 μm, out of which SBS flowed at 3.33, 7.67, and 16.67 μL/s for 5 s. Calculation of shear stress (τ) was done using the Hagen-Poiseuille equation (τ = 4 μQ/πR³), where μ is dynamic viscosity, Q is flow rate, and R is radius of the capillary tube [28 (link),42 (link)].

Cells were loaded with 10 μM Fura2-AM (Dojindo, Kumamoto, Japan) in SBS for 30 min at 24–26 °C, superfused with SBS for 10 min, and Fura-2 fluorescence signals were measured at 0.1 Hz using the Argus/HisCa imaging system (Hamamatsu Photonics, Hamamatsu, Japan) driven by Imagework Bench 6.0 (INDEC Medical Systems, Santa Clara, CA, USA). In each analysis, the whole cell area was chosen as the region of interest to average the fluorescence ratio.

Untreated monolayer cells and SMG-ECM-treated cell aggregates and monolayer cells were incubated with 2 μM Fura-2AM (Millipore, cat. # 344905, Billerica, MA) at 37 °C for 45 min and then washed 2 times with Ca²⁺-free SES buffer (i.e., Standard External Solution, containing 10 mM HEPES, 120 mM NaCl, 5.4 mM KCl, 1 mM MgCl₂, and 10 mM glucose, pH 7.4). For assay, Fura-2 fluorescence intensity of the loaded control cells was monitored with a CCD camera-based imaging system (Hamamatsu Photonics, Japan) mounted on an Olympus XL70 inverted microscope equipped with an Olympus 40× (1.3 NA) objective. Fura-2 dual excitation and emission were accomplished using 340- and 380-nm excitation filters and a 510-nm emission filter. Imaging data acquisition was accomplished using MetaFluor software (Molecular Devices, San Jose, CA). Fluorescence traces show intracellular calcium [Ca2+]i values from an average of at least 50 or more cells and are representative of results obtained in at least 3 individual experiments.