Mc3t3 e1 cells

The NR8383 cell line was purchased from Shanghai Institute for Biological Sciences, Chinese Academy of Science. NR8383 cells were cultured at 37 °C in F12 K medium (Sigma-Aldrich, St. Louis, MO) supplemented with 15% FBS (PAA Laboratories GmbH, Austria), 2 mM L-glutamine (Amresco Inc., USA), and 1% penicillin/ streptomycin (Haoyang Biological Manufacture Co., Tianjin, China) in an incubator with 5% CO₂ [23 (link)]. MC3T3-E1 cells (iCell Bioscience Inc., Shanghai, China) were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma-Aldrich, St. Louis, MO) supplemented with 10% FBS in an incubator with 5% CO₂.
Cell viability was determined by Cell Counting Kit-8 (CCK-8, Beyotime Institute of Biotechnology, China). Briefly, NR8383 (1 × 10⁷ cells/mL) or MC3T3-E1 cells (1 × 10⁵ cells/mL) in 96-well plates were exposed to different concentrations of AG-QDs (0, 10, 25, 50, 100, 200, and 500 μg/mL) at 37 °C for 12, 24, 48, 72 and 96 h, and were then treated with CCK-8 probes to determine cell viability with a microplate reader (Thermo-1500, USA).

Cell cultures were conducted as previously described [18 (link)]. MC3T3-E1 murine osteoblastic cells and MCF-7 human breast cancer cells were purchased from the American Type Culture Collection (Manassas, VA, USA). MC3T3-E1 cells were seeded in T75 flasks, maintained at 37 °C with 5% CO₂, and cultured in the alpha-minimum essential medium (Gibco, Baithersburg, MD, USA) containing 1% penicillin/streptomycin (Gibco) and 10% fetal bovine serum (Gibco). For osteoblast differentiation, MC3T3-E1 cells were differentiated using osteoblastic differentiation medium (DM), which was prepared by adding 10 mM β-glycerophosphate and 50 μg mL⁻¹ L-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) to the culture medium. Cells treated with DM for six days were used for ALP activity, protein, and mRNA analyses. MCF-7 cells were cultured in MEM containing 10% fetal bovine serum and 1% penicillin streptomycin in a 5% CO₂ incubator.

Murine osteoblastic MC3T3-E1 cells were purchased from the Committee of Type Culture Collection (Chinese Academy of Sciences, Beijing, China). All in vitro studies were performed using passages 5–10. In this study, MC3T3-E1 cells were seeded in 25 cm² flat-bottom cell culture flasks and supplemented with α-MEM (Gibco, Life Technologies, California, USA) containing 10% fetal bovine serum (Gibco, Life Technologies, California, USA) in a humidified atmosphere with 5% CO₂ at 37 °C. In general, the cells were subcultured when they reached a subconfluent state after 3 days of culturing.

MC3T3-E1 cells (RCB1126, an osteoblast-like cell line from C57BL/6 mouse calvaria) were obtained from the RIKEN Cell Bank (Tsukuba, Japan). MC3T3-E1 cells were cultured at 37°C in 5% CO₂ atmosphere in α-modified minimal essential medium (α-MEM; GIBCO). Unless otherwise specified, the medium contained 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin.

MC3T3‐E1 cells, a mouse calvarial osteoblast cell line, were obtained from Riken Bio Resource Center (Tsukuba, Japan). MC3T3‐E1 cells were maintained in α‐minimal essential medium (12571‐063; Gibco‐BRL, Grand, Island, NY, USA) supplemented with 10% FBS (Biosera Nuaillé, France) and 100 U·mL⁻¹ penicillin–streptomycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Osteoblast differentiation was induced by culturing cells in an osteogenic medium containing 50 μg·mL⁻¹ ascorbic acid (FUJIFILM Wako Pure Chemical Corporation) and 10 mm β‐glycerophosphate (Sigma‐Aldrich, St. Louis, MO, USA) for 7 days. Cells were treated with 1, or 10 μm clodronate (Sigma‐Aldrich). Ionomycin (AdipoGen Life Sciences, San Diego, CA, USA) treatments were performed at 1 μm. Brefeldin A (Cayman Chemical, Ann Arbor, MI, USA) was used at 10 μm. HEK293T cells (Takara Bio, Shiga, Japan) were cultured in Dulbecco's modified Eagle's medium (DMEM) (12320‐032; Gibco‐BRL) supplemented with 10% FBS (Biosera Nuaillé) and 100 U·mL⁻¹ penicillin–streptomycin (FUJIFILM Wako Pure Chemical Corporation).