Basic research software

BMMs were cultured at a concentration of 1× 10⁴ cells per well in 48-well trays with complete α-MEM nutritional solution. Next day, 10 µM asiatic acid with 50 ng/ml GST-rRANKL and 25 ng/ml M-CSF was added and cells was incubated for one day. After washed by HANKS balanced salt solution (supplemented with 1 mM probenecid and 1% FBS), the cells were stained by 4 mM Fluo4 solution (Fluo4-AM dissolved in 20% pluronic-F127) (Molecular probes, Thermo Fisher Scientific, Scoresby, Australia) at 37°C for 45 min. Staining solution was removed and cells were maintained in HANKS solution at room temperature for 20 min. Cells were washed prior to fluorescence signals being detected by a fluorescent microscope at a wavelength of 488 nm. Continued images were recorded every 3 seconds for 1 min and Ca²⁺ flux quantified by a Nikon Basic Research Software. Calcium oscillation intensity was calculated and presented as the interval between the maximum and minimum of fluorescence value within the scopes.

The calcium oscillation was estimated by a Fluo4‐AM kit (Thermo Fisher Scientific, Waltham, MA, USA). The BMMs seeded at 1 × 10⁴ cells per well were cultured with or without 1 μM Rob in the presence of rm‐sRANKL and M‐CSF for 24 h. The RANKL‐induced osteoclasts were rinsed twice with Hanks' Balanced Salt solution (HBSS) supplemented with 1 mM probenecid as well as 1% FBS, and cultured with 100μL/well Fluo4 solution for 45 min at 37°C. After staining was complete, the cells were washed and kept at room temperature for 20 min. The intracellular free calcium, visualized as fluorescence at different densities, was detected using a fluorescence microscope at 488 mm (excitation wavelength). Images were obtained every 2 s for 1 min. Cells with more than two oscillations were counted as oscillating cells, and their amplitudes were measured using Nikon Basic Research Software.

BMMs (1×10⁴ cells/well) were cultured in a 48-well plate with or without treatments according to different groups. In the negative control group, cells were incubated only with M-CSF (50 ng/ml). In the positive control group, cells were incubated with 50 ng/ml M-CSF and RANKL in the absence of NOT. While in the treatment group, cells were dealt with 50 ng/ml M-CSF and RANKL as well as indicated concentration of NOT (10 μM). After 24 h, the cells were rinsed three times with assay buffer (HANKS balanced salt solution added with 1 mM probenecid and 1% FBS), and incubated with Fluo-4 staining solution (Fluo4-AM dissolved in 20% (w/v) pluronic-F127in DMSO diluted in assay buffer) in the dark room at 37°C for 45 min. When staining was completed, the cells were washed once in assay buffer. The fluorescence intensity was observed with fluorescent light (at an excitation wavelength of 488 nm) by inverted fluorescence microscope (Nikon). Images were acquired every 2 s for 3 min. Cells with at least two oscillations were regarded as oscillating cells. The average amplitude of individual oscillating cell was analyzed by Nikon Basic Research Software (Nikon).

BMMCs cells were isolated and cultured on the 48‐well plates (2 × 10⁴ cell/well) with M‐CSF (50 ng/mL) for 24 hours. Then, cells were induced in the presence of M‐CSF (50 ng/mL) with or without RANKL (50 ng/mL) and treated with NBIF (8 μM) for the next 48 hours. After washing with assay buffer, cells were added with 4 μM Fluo4 staining solution. The cytoplasmic Ca²⁺ was examined by inverted fluorescence microscope (Nikon Ti‐U) at 488 nm, and the results were analysed using Nikon Basic Research Software. After scanned and plotted at the interval of 2 seconds for 3 minutes, cell images recorded with 2 or more peaks were recorded as oscillating. Moreover, the fluorescence intensities between the highest and lowest group within the oscillating area were detected.

BMMs (1.5 × 10⁴) were seeded in 48‐well plates, with added components for different groups. In the treatment group, the cells were treated with GST‐rRANKL (50 ng/mL), M‐CSF (50 ng/mL) and astilbin (10 μmol/L); in the positive control group, cells were treated with GST‐rRANKL (50 ng/mL) and M‐CSF (50 ng/mL) but were not exposed to astilbin; and in the control group, the cells were treated with M‐CSF alone (50 ng/mL). After cultured for 24 hours, the cells were washed twice with assay buffer (Hank's balanced salt solution with 1 mmol/L probenecid and 2% foetal calf serum (FCS)) and stained with Fluo4 staining solution (Fluo4‐AM dissolved in 20% (w/v) pluronic‐F127 in dimethyl sulfoxide (DMSO) added to assay buffer) in the dark at 37°C for 45 minutes. When staining was completed, the cells were rinsed again with assay buffer and incubated on a bench in the dark for 20 minutes after removing the staining solution. The intensity of fluorescence was observed under fluorescent light (at an excitation wavelength of 488 nm) by an inverted fluorescence microscope (Nikon, Tokyo, Japan). Images were captured every 2 seconds for 4 minutes. Cells with at least two oscillations were counted as oscillating cells. The average amplitude of each oscillating cell was analysed by Nikon Basic Research Software as previously described.22

BMMs (3 × 10⁴) were seeded into a 48‐well plate with treatments added based on different groups. In the treatment group, cells were treated with GST‐rRANKL (50 ng/mL), M‐CSF (50 ng/mL), and MA (10 μmol L⁻¹); in the positive control group, cells were treated with RANKL (50 ng/mL) and M‐CSF (50 ng/mL) but without MA; and in the control group, cells were treated only with M‐CSF (50 ng/mL). After being cultured for 24 h, the cells were washed twice with Assay buffer (HANKS buffer dissolved with 1 mmol L⁻¹ probenecid and 2% FCS) and stained with Fluo‐4 staining solution (Fluo‐4 AM dissolved in 20% (w/v) Pluronic F‐127 in DMSO added to Assay buffer) in the dark at 37°C for 45 min. When the staining was finished, the cells were rinsed again with Assay buffer and incubated at room temperature in the dark for 20 min after removal of the staining solution. The intensity of fluorescence was observed under fluorescent light (at an excitation wavelength of 488 nm) by inverted fluorescence microscope (Nikon). Images were captured every 2 s for 4 min. Cells with at least two oscillations were counted as oscillating cells. The average amplitude of each oscillating cell was analysed by Nikon Basic Research Software (Nikon).