Von kossa stain kit

Primary bone-marrow cells were isolated from femurs and mixed with RBC lysis buffer (11814389001; Sigma-Aldrich, Merck KGA, Darmstadt, Germany) to collect mononuclear cells. Upon incubating mononuclear cells in medium (DMEM with 10% fetal bovine serum; Gibco) overnight, adherent cells were harvested and incubated (10⁵ cells/well, 24-well plates) in osteogenic medium (StemPro™ Osteogenesis Differentiation Kit; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 18 days. Mineralized nodule formation was investigated using von Kossa Stain Kits (ab150687; Abcam Cambridge, UK), according to maker’s instruction. Von Kossa-stained areas in each field (mm²/filed) was measured using light microscope and image analyzer.

We assessed the impacts of miR-29a-AS and pre-miR-29a on the osteogenic potentials of the harvested primary bone marrow mesenchymal cells. Briefly, primary bone marrow mesenchymal cells harvested from femurs were mixed with red blood cell lysis buffer (11814389001; Sigma-Aldrich, St. Louis, MO, USA) to isolate mononuclear cells. Upon incubating mononuclear cells in Dulbecco’s modified Eagle medium with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA) overnight, adherent cells were collected and incubated in osteogenic medium (10⁵ cells/well, 24-well plates) (StemPro™ Osteogenesis Differentiation Kit; Thermo Fisher Scientific, Waltham, MA, USA) for 18 days. The extent of mineralization was assessed employing von Kossa Stain Kits (ab150687; Abcam, Cambridge, UK), following instructor’s protocol. Calcium in mass deposits would be stained with black color by the Kit, and the black color-stained area (mm²/filed) of von Kossa-stained mineralized matrices in each ×125 magnification field were measured applying light microscopy (Zeiss Image Analysis System, Oberkochen, Baden-Württemberg, Germany) [19 (link),21 (link)].

BMSCs were harvested from the mouse femur as previously described (Wu et al. 2021 (link); Ma et al. 2024 (link); Xie et al. 2023 (link); Li et al. 2023 (link)). Harvested primary BMSCs were mixed with red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) to isolate mononuclear cells. Following overnight incubation of mononuclear cells in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA, USA), adherent cells were harvested. These cells were subsequently cultured in osteogenic medium (10⁵ cells/well in 24-well plates) (StemPro™ Osteogenesis Differentiation Kit; Thermo Fisher Scientific, Waltham, MA, USA) for passaging every 5–7 days, maintaining a plating density of 100 cells/cm² for around 18 days. The extent of mineralization was assessed employing von Kossa Stain Kits (Abcam, Cambridge, UK), following instructor’s protocol. Calcium mass deposits were stained black by the kit, and the area of von Kossa-stained mineralized matrices (mm²/field) in each × 125 magnification field was measured using light microscopy and image analysis software (Carl Zeiss). Six fields were randomly selected from each well, with a total of six wells for each treatment, to quantify the von Kossa-stained area (Ko et al. 2023a (link)).

Mesenchymal cells and macrophage precursor cells in bone marrow were isolated, as previously described [24 (link)]. Bone-marrow stromal cells (10⁵ cells/well, 24-well plates) were seeded in an osteogenic condition using StemProTM Osteogenesis Differentiation Kits (A1007201 Thermo Fisher Scientific Inc., Waltham, MA, USA) and in an adipogenic condition using StemProTM Adipogenic Differentiation Kits (A1007001) for 21 days and 15 days, respectively. Mineralized matrices and adipocytes were stained using von Kossa stain kits and Nile Red stain kits (Abcam, Cambridge, UK). Bone-marrow macrophage precursor cells (5 × 10⁴ cells/well, 48-well plates) were incubated in αMEM with 15 ng/mL M-CSF and 40 ng/mL RANKL (R&D Systems, Minneapolis, MN, USA) for 1 week. Osteoclasts were probed using TRAP stain kits. Von-Koss-stained mineralized matrices in each low-power field (x125 magnification) and Nile-red-stained adipocytes and TRAP-stained osteoclasts in each high-power field were measured using the Zeiss Image Analysis System.