Dm5500

Brains hemispheres were harvested from anaesthetized mice and immediately frozen in isopentane. 8-μm cryosections were collected and fixed with 4% paraformaldehyde. For immunostainings, sections were stained with 0,01% Thioflavin S (Sigma) for 15min at room temperature, and after washes in ethanol and PBS, stained with Hoechst (Life Technology). After washing in PBS, tissue sections were mounted with Dako mounting medium. Images were acquired using Leica DM 5500 (Leica Microsystems) CMOS camera 2900 Color at the same exposure time. Quantitative analysis of the immunofluorescence data was carried out by histogram analysis of the fluorescence intensity at each pixel across the images using Image J (Fiji; National Institutes of Health). Appropriate thresholding was employed to all the images of each single experiment to eliminate background signal in the images before histogram analysis. Fluorescence intensity and signal positive areas were calculated using the integrated “analyse particles” tool of the Fiji software, and statistical analysis were performed using Prism 6 (GraphPad Software).

To induce osteoclasts, RAW264.7 cells (5000 cells/cm²) were cultured in the presence of receptor activator of nuclear factor kappa-Β ligand (RANKL, 20 ng/ml), Mg (0 or 10 mM) and VC (0 or 50 μg/ml) for 4 days. Then quantitative real-time PCR (qRT-PCR) analysis and tartrate-resistant acid phosphatase (TRAP) staining (cat# 387, sigma) were conducted. TRAP-positive multinucleated cells with more than three nuclei were counted as osteoclasts under a light microscope. To further study the Mg effect on osteoclastogenesis induced by LPS or RANKL, RAW264.7 cells (5000 cells/cm²) were cultured with LPS (100 ng/ml) or RANKL (20 ng/ml) plus Mg (0 or 10 mM) for 4 days for TRAP staining and qRT-PCR, and for 6 days for bone resorption function testing with the medium changed every 3 days. Osteo Assay Surface 24-well Multiple Well Plate (Corning, Product Number 3987) was used for testing the resorption function with pit formation assay. After cell culture for 6 days, the cells on the plate were lysed by 4% NaClO and the plate was washed with deionized water. The pit area in each plate was imaged (Leica DM5500; Leica Micro-systems, Wetzlar, Germany) and analyzed by ImageJ (version 1.51, Wayne Rasband, National Institute of Heath, USA).

Tetrazolium (MTT) method and Live/Dead staining were performed to evaluate the effect of Mg and VC on the viability of MC3T3-E1 cells. For MTT test, the cells were seeded onto 96-well plates at a density of 2 × 10³ cells/well. After overnight incubation, the cells were treated with different concentrations of methylprednisolone (MPS) or lipopolysaccharide (LPS) with/without Mg and VC supplemented for 24 h. Then cells were incubated with 50  μl (1 mg/ml) MTT solution at 37 °C in dark for 4 h. After discarding the MTT solution, 50  μl DMSO was added to each well, and the absorbance at 575 nm was detected. For Live/Dead staining, the cells were seeded onto 4-well chamber slides at a density of 1 × 10⁴ cells/well. After overnight incubation, the cells were treated with different concentrations of MPS with/without Mg and VC supplemented for 24 h. Then cells were stained with live/dead cell imaging kit (488/570) (R37601, Invitrogen) and analyzed under a fluorescence microscope (Leica DM5500; Leica Micro-systems, Wetzlar, Germany).