Cell dissociation solution

SHSY5Y neuroblastoma cells (ATCC, USA) which are growing as a mixture of floating and adherent cell types were cultured in DMEM (Gibco BRL, USA) media containing 10% heat-inactivated fetal bovine serum (Gibco BRL, USA) and 1% penicillin/streptomycin (Invitrogen, USA) in a T75 flask and were subcultured every 3 days. The cells were routinely washed with phosphate-buffered saline (PBS, Welgene, South Korea) and detached using cell dissociation solution (Sigma-Aldrich, USA). To observe cell growth on various materials, sterilized materials were placed into each well of a 12-well plate (Nunc, Thermo Scientific, USA), and 1 × 10⁴cells in 2 ml of media were loaded per well. The plate was incubated at 37 °C, 5% CO₂ for 24 h. Cells were then collected and counted using a haemocytometer counting chamber (Marienfeld, Germany) after mixing with trypan blue solution (Sigma-Aldrich, USA) at a 1:1 ratio. For cell viability, the ratio of live cell (%) was calculated by dividing total cell number by live cell number. The statistical test was performed by two-tailed student’s t-test for each group against control (*p < 0.05, n = 5 for empty, glass, agar, PET, and n = 8 for PDMS, Silicone. n = number of well). The cells were imaged via phase contrast optical microscopy (AF6000B, Leica, Germany).

Osteoclast function was assessed using a hydroxyapatite resorption assay. BMMs were seeded at a concentration of 1 × 10⁵ cells/well in 6-well collagen-coated culture plates in complete α-MEM with M-CSF overnight. Osteoclast formation was then stimulated through the addition of 50 ng/mL GST-rRANKL for 4 days. Once mature osteoclasts were observed cells were gently removed from the wells using cell dissociation solution (Sigma, St. Louis, MO, USA). Mature osteoclasts were then plated onto hydroxyapatite-coated plates (Corning, New York, NY, USA) in equal numbers and treated with Bajijiasu at various concentrations (0.4 and 0.8 mM), in the presence of 50 ng/mL GST-rRANKL and M-CSF for 48 h. Half of the wells were dried for hydroxyapatite resorption visualisation using a Nikon microscope (Nikon Corporation, Tokyo, Japan), while the remaining wells were fixed and stained for TRAcP activity for osteoclast counting. The percentage of hydroxyapatite resorption areas were measured using Image J software and normalized to the number of osteoclasts (National Institutes of Health, Bethesda, MD, USA).

MDM were challenged with GFP-375^SR as described and cells were harvested using Cell Dissociation Solution (Sigma) for 20 min at 37°C. The cell suspension was recovered and centrifuged to generate a cell pellet. The cell pellet was resuspended in PBS and streaked onto PolyFrost Microslides (Solmedia, Shrewsbury, UK). Once dry, slides were fixed using 4% paraformaldehyde (PFA) for 15 min. Excess PFA was removed and slides were washed in PBS and left until completely dried. Once dry, 25 μl Vectashield^® Mounting Medium solution containing DAPI nuclear stain (1.5 μg/ml) (Vector Laboratories, Inc. Burlingame, CA) was added to each spot and a glass coverslip mounted on top. Slides were visualised using Axioscope KS400 fluorescence microscope using Carl Zeiss Axioscope 3.0 software.

A resorption pit assay was used to evaluate osteoclast function. BMMs were seeded into 6‐well plates at a density of 1 × 10⁵ cell/well and stimulated with 20 ng/ml M‐CSF for 3 days and then treated with 20 ng/ml M‐CSF and 50 ng/ml RANKL for 5 days until mature osteoclasts formed. Detached the Cells from the wells using a cell dissociation solution (Sigma, St. Louis, MO, USA) and then plated into 48‐well plates with bone slices. The mature osteoclasts were treated with different concentrations of tetrandrine in the presence of M‐CSF (20 ng/ml) and RANKL (50 ng/ml). After 48 h, bone slices were stained with Toluidine Blue to detect resorption pits. Use Image J software (NIH, Bethesda, MD, USA) to analyze the percentage of resorption areas of bone slices.

293 FT cells were seeded in 6-cm dishes 24 hours prior to transfection. The cells were transfected with pXJ3′ empty vector, pXJ3′-S and pXJ3′-S-D1128A plasmids using Lipofectamine 2000 reagent (Invitrogen) according to manufacturer’s protocol and harvested at 72 hours post-transfection. The cells were first detached using the cell dissociation solution (Sigma), washed twice in 1x PBS and incubated with purified mouse mAb 7G12 [31] (link) (that binds to the S1 subunit) in 1x PBS containing 1% bovine serum albumin (BSA) for 3 hours at 4°C on a nutator. The cells were washed 3 times using 1x PBS containing 1% BSA and then incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Santa Cruz) secondary antibody for 1 hour at 4°C on the nutator. Cells were washed again 3 times and immediately used for FACS analysis using the CyAn flow cytometer (Beckman Coulter). All FACS data was analysed using the FlowJo software application.