Osteoimage mineralization assay kit

The formation of mineralized matrix in vitro was quantified using an OsteoImage mineralization assay kit according to manufacturer's instructions (Cat. number PA-1503; Lonza, USA). Briefly, culture medium was removed, and cells were washed once with PBS and then fixed with 70% cold ethanol for 20 minutes. Next, diluted staining reagent was added at a level recommended by the manufacturer, and plates were incubated in the dark for 30 minutes at room temperature. The cells were then washed, and staining was quantified using a fluorescence plate reader (Molecular Devices Co., Sunnyvale, CA, USA) with excitation and emission wavelengths of 492 and 520 nm, respectively.

P2-MSCs were detached, re-plated at 20.000 cells/cm² in 24-well TC-treated plates, and grown to confluence in MSC expansion medium. To induce osteogenesis, medium was replaced by StemMACS™ OsteoDiff Medium (Miltenyi Biotec) and cultures maintained for 21 days (medium changed weekly). Control plates were set up in MSC expansion medium. Calcium deposits were revealed by OsteoImage™ Mineralization assay kit (Lonza, Basel, Switzerland) according to manufacturer. Pictures were taken using an inverted fluorescence DM IRB Leica microscope (Leica) equipped with LAS image acquisition software (Leica). Image analysis was performed by QWin image analysis software (Leica) to quantify the percentage of fluorescent (mineralized) areas. Two-tailed unpaired t-test was performed.

For osteogenesis, a total of 15,000 cells/well were plated in complete medium in 48-well cell culture plates. At 80% confluence, cells were provided with osteogenic medium [α-MEM supplemented with FBS (10%), ascorbate-2-phosphate (50 µM), β-glycerophosphate (10 mM), dexamethasone (100 nM), penicillin–streptomycin (100 U/mL and 100 μg/mL), and amphotericin-B (2.5 μg/mL)], along with three different concentrations of Mg²⁺ (Fluka, Buchs, Switzerland) and two concentrations of 1,25D (Sigma Aldrich, Taufkirchen, Germany), as shown in Table 1. The cells were kept in osteogenic medium for 21 days and the commercially available OsteoImage Mineralization Assay kit (Lonza, Walkersville, MD, USA) was used to determine the development of hydroxyapatite material formation around cells, according to the manufacturer’s instructions. The EnSpire Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) was used to quantify hydroxyapatite formation at excitation/emission wavelengths of 475/530 nm. To obtain the mineralization/nuclei ratio, the fluorescence of DAPI was measured at 358/461 nm and the hydroxyapatite readout was divided by the DAPI nuclear readout (mineralization/nuclei ratio) to correct for varying cell density.

After 15 days of osteogenic stimulation, JPCs were fixed with fixation buffer (Biolegend, California, USA) and stained with 40 mM Alizarin red solution (pH 4.2, Sigma-Aldrich) for 20 min. Unbound dye was removed by washing with deionized water and images were taken using an inverted microscope (Leica, Wetzlar, Germany). The stained plates were quantified using alizarin red S staining quantification assay kit (ScienCell, California, USA) following the manufacturer’s instructions and photometrical quantification of the alizarin staining was performed at a wavelength of 405 nm using a microplate reader (Biotek, Bad Friedrichshall, Germany). JPCs were further stained for hydroxyapatite detection using the OsteoImage mineralization assay kit (Lonza, Basel, Switzerland) following the manufacturer’s instructions. Images were taken using an Axio Observer Z1 fluorescence microscope (Zeiss, Oberkochen, Germany).

Nontransduced or lentiviral‐transduced DDCs were seeded in 12‐well plates at a density of 2 × 105 cells/well in osteogenic differentiation medium (αMEM containing 10% FBS, 2mM L‐glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 10mM β‐glycerol phosphate, 50 µM ascorbic acid, 10nM dexamethasone) for up to 14 days with fresh medium changes every 3 days. To examine matrix mineralization at day 14, DDCs were fixed in 4% paraformaldehyde and stained for 30 min with 1% Alizarin Red ethanol solution. Alkaline phosphatase activity was monitored in day 14 paraformaldehyde‐fixed DDC cultures following treatment with 1‐step 4‐nitro blue tetrazolium/5‐bromo‐4‐chloro‐3‐indolyl phosphate substrate solution (Thermo Fisher Scientific, Waltham, MA, USA) for 15 min at 37°C in a dark humidified chamber. Bone‐specific matrix hydroxyapatite was detected in day 14 cultures using the OsteoImage Mineralization Assay Kit (Lonza Group, Basel, Switzerland) following the manufacturer's protocol. Fluorescence microscopy was carried out for semiquantitative analysis of hydroxyapatite levels. Data were quantified on a fluorescent plate reader set to the appropriate excitation/emission wavelengths (492 nm/520 nm).

P2-MSCs cultured in chamber slides were induced to terminal differentiation into adipocytes using StemMACS® AdipoDiff Medium (Miltenyi Biotech) or into osteocytes by StemMACS® OsteoDiff Medium (Miltenyi Biotech). Media were refreshed every 48 h and cultures were maintained for 21 days.
To detect lipid droplet accumulation, the medium was removed, wells were washed twice with prewarmed D-PBS, and cells were incubated in 200 nM Nile Red (Thermo Scientific) for 10 min at 37 °C in the dark. Calcium deposits were revealed by OsteoImage™ Mineralization assay kit (Lonza) according to the manufacturer’s instructions. Pictures were taken using an inverted fluorescence DM IRB Leica microscope (Leica), equipped with LAS image acquisition software (Leica).

The in vitro formed mineralized matrix was quantified using the Osteo-Image Mineralization Assay Kit (Lonza, Walkersville, MD, USA, Cat. No. PA-1503). Culture media were removed and cells washed once with PBS, and fixed with 70% cold ethanol for 20 min. Appropriate amount as recommended by the manufacturer of diluted staining reagent was added, and plates were incubated in dark for 30 min at room temperature. Cells were washed, and staining quantitation was performed using fluorescent plate reader at 492/520 excitation emission wavelengths.