Argus hisca imaging system

The change in [Zn²⁺]_i was measured using FluoZin-3 (35 (link)). Cells were incubated with 10 μM FluoZin-3AM for 30-min in SBS at room temperature. Upon excitation at a wavelength of 488 nm, FluoZin-3 fluorescence signals emitted at wavelengths longer than 510 nm were collected at 0.1 Hz using the Argus/HiSCA imaging system (Hamamatsu Photonics, https://www.hamamatsu.com/jp/en.html) driven by Imagework Bench v6.0 (INDEC Medical Systems, http://www.indecmedical.com/). Changes in fluorescence intensity were calculated as Zn²⁺_i (F/F₀) by the normalization at time zero. For each analysis, fluorescence signals were averaged over the whole-cell area. To quantitatively measure changes in Zn²⁺ levels, responses from 50 cells within a single coverslip were averaged, and this protocol was replicated to change the coverslip. To measure changes in intracellular Ca²⁺ ([Ca²⁺]_i), HEK cells, which were loaded with 10 μM Fura2-AM (DOJINDO) in SBS for 30 min at room temperature, were superfused with SBS for 10 min to remove residual Fura2-AM in the recording chamber. Fura2 fluorescence signals, elicited upon excitation at wavelengths of 340 nm and 380 nm, were recorded at 0.1 Hz. Changes in the fluorescence intensity ratio were calculated as Ca²⁺_i (F₃₄₀/F₃₈₀) and analyzed in the similar manner to [Zn²⁺]_i.

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)].

HEK, FLSs, and DRG cells, which were loaded with 10 μM Fura2-AM (Dojindo, Kumamoto, Japan) in SBS for 30 min at room temperature, were 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). Since the efficacy of gene transfection in HEK cells and the TRPA1 expression level in FLSs and DRG cells were similar but not identical from cell to cell, we collected 50, 5–11, and 8–12 single cells of HEK, FLSs, and DRG cells, respectively, on one coverslip to obtain the average response. We repeated the same protocol with other coverslips to obtain the mean and standard error of the mean (SEM) of independent experiments. In each analysis, the whole cell area was chosen as the region of interest to average the fluorescence ratio.