Calcium phosphate (CaP) deposition was detected by Calcein (C30H26N2O13), a fluorescent chromophore that binds specifically to Ca2+ in cultured iPSC-VSMCs (Du et al., 2001). Cultured cells were washed once with PBS and fixed in chilled (4 °C) 4% paraformaldehyde (PFA) for 30 min. Cells were then incubated for 30 min in the dark with Calcein staining solution (Sigma, #C0875), washed three times with 50 mM TBS (pH 9) (each for 3 min), and then counterstained with DAPI for 10 min in the dark. Cells were covered with 400 μL of 50 mM TBS (pH 9), and Calcein fluorescence was detected using a BZ-9000 BioRevoTM fluorescence microscope (Keyence, Neu-Isenburg, Germany) immediately after staining. Alizarin Red S (ARS) staining was performed using the osteogenesis quantitation kit (Merck Millipore, # ECM815), following the manufacturers’ instructions. Stained cells were imaged using the BZ-9000 BioRevo™ fluorescence microscope (Keyence, Neu-Isenburg, Germany).
Osteogenesis quantitation kit
Manufactured by Merck Group
Sourced in United States
The Osteogenesis Quantitation Kit is a laboratory tool designed to quantify the extent of osteogenesis, or bone formation, in cell culture experiments. It provides a standardized method to measure the level of osteogenic differentiation of cells.
Automatically generated - may contain errors
Lab products found in correlation
Phenol red, by Thermo Fisher Scientific (2 mentions)
Bz9000 biorevo fluorescence microscope, by Keyence (1 mentions)
Recombinant human tgf β1, by Thermo Fisher Scientific (1 mentions)
Live dead viability cytotoxicity kit, by Thermo Fisher Scientific (1 mentions)
Mab1959, by Merck Group (1 mentions)
Activin b, by R&D Systems (1 mentions)
Nhost cells, by Lonza (1 mentions)
Oil red o, by Merck Group (1 mentions)
Eclipse 50i microscope, by Nikon (1 mentions)
Alizarin red s staining solution, by Merck Group (1 mentions)
Nile red, by Merck Group (1 mentions)
Kinetix camera, by Teledyne (1 mentions)
13 protocols using osteogenesis quantitation kit
1
Calcium Phosphate Deposition Analysis
2
Dental Pulp Stem Cell Adhesion and Differentiation
3
Quantifying Mineralization via Alizarin Red
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Staining of the cells was performed with the Osteogenesis Quantitation Kit (Merck KGaA). Pictures were taken to monitor mineralization. To quantify mineral deposition, incorporated alizarin red was solubilized and absorbance was compared with a standard curve of alizarin red concentrations (0–2 mM). Alizarin red data are expressed as mM.
4
Activin A Regulation of Osteoblast Mineralization
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Example 5
Osteoblast Mineralization of Normal Human Osteoblast Cells.
Normal human osteoblast (NHOst) cells (Lonza Walkersville) were cultured in Minimal Essential Medium Alpha (MEM-α) without phenol red (Invitrogen), 10% fetal bovine serum (FBS), 5 units penicillin-streptomycin and 0.25 μg/ml amphotericin B. Mineralization experiments were performed in 12 well plates seeded at 2.5×104 cells/well using cells between passages 3 and 6. Mineralization media was the same as above except for the addition of 2% heat-inactivated, charcoal-stripped FBS, 20 mM HEPES and 1.8 mM CaCl2. Media was supplemented with 100 nM dexamethasone and 10 nM β-glycerol phosphate (Dex βGP)±50 ng/ml activin A or ±10 ug/ml anti-activin A (Anti-ActA; Cat No. MAB3381, R&D Systems). Differentiation media was changed every 2-3 days over a 14 day period. NHOst cells were analyzed for bone mineral deposition using an osteogenesis quantitation kit (Chemicon). Cells were rinsed with PBS, fixed in formalin free fixative and stained with alizarin red.
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5
Osteoblast Mineralization Modulation
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Example 7
Osteoblast Mineralization of Normal Human Osteoblast Cells.
Normal human osteoblast (NHOst) cells (Lonza Walkersville) were cultured in Minimal Essential Medium Alpha (MEM-α) without phenol red (Invitrogen), 10% fetal bovine serum (SAFC Biosciences), 5 units penicillin-streptomycin, and 0.25 μg/ml amphotericin B. Mineralization experiments were performed in 12-well plates seeded at 5×104 cells/well using cells between passages 3 and 6. Mineralization media was the same as above except for the addition of 10% heat-inactivated, charcoal-stripped fetal bovine serum, 20 mM HEPES, and 1.8 mM CaCl2. Media was supplemented with 100 nM dexamethasone and 10 nM β-glycerol phosphate (Dex βGP)±50 ng/ml activin B (R&D Systems) and ±20 μg/ml ActRIIa-mFc. Differentiation media was changed every 2-3 days over a 14-day period. NHOst cells were analyzed for bone mineral deposition using an osteogenesis quantitation kit (Chemicon). Cells were rinsed with PBS, fixed in formalin-free fixative, and stained with alizarin red.
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6
Quantitative Alizarin Red Staining
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Calcium deposits were determined by the Osteogenesis Quantitation Kit (Sigma-Aldrich, USA). Briefly, cells were washed with PBS twice. Then cells were fixed with 10% formaldehyde and incubating at room temperature for 15 min. Subsequently, cells were washed with distilled water for 3 times and stained with 1x Alizarin Red at room temperature for 20 min. After acid extraction, extracted solution was measured at 405 nm. A serial dilution of ARS standards was prepared for quantitative analysis according to the manufacturer’s instructions, and measurements were performed in triplicate.
7
Quantification of Calcium Deposition
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Calcium deposits were determined by the Osteogenesis Quantitation Kit (Sigma-Aldrich, USA). Briefly, Cells were washed with PBS twice. Then cells were fixed with 10% formaldehyde and incubating at room temperature for 15 min. Subsequently, cells were washed with distilled water for 3 times and stained with 1x Alizarin Red at room temperature for 20 min. After acid extraction, extracted solution was measured at 405 nm. A serial dilution of ARS standards was prepared for quantitative analysis according manufacturer's instructions, measurements were performed in triplicate.
8
CRP Enhances Bone Matrix Production
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Example 3
The effect of CRP on bone matrix production was assayed in the chondrocyte preosteoblast cell line, MC3T3-E1. The cells were differentiated according to the method provided with the Millipore Osteogenesis kit and then treated with CRP at 1 or 10 μM for 48 hours. Bone matrix deposition was assayed according to the Alizarin Red S staining method provided with the Millipore Osteogenesis Quantitation kit. Relative to a standard curve generated using Alizarin dye, CRP-treated cells showed a dose-dependent increase in Alizarin Red-detectable calcium indicating a four to fivefold increase in bone matrix deposition. CRP increased the production of bone matrix in both differentiating and terminally differentiated cells.
9
Staining Techniques for Cellular Differentiation
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Cell cultures were stained with Oil Red O (Millipore) to determine the efficiency of adipogenic differentiation. Cells were fixed with 4% paraformaldehyde for 30 min. They were then stained with 0.4% Oil Red O in 60% isopropanol for 1 h.
Alizarin Red was used to estimate the efficiency of osteogenic differentiation. Cells were fixed with 10% paraformaldehyde for 30 min. They were then stained using the Osteogenesis Quantitation Kit (Millipore) following the manufacturer’s instructions.
Toluidine blue staining was used to visualize chondrogenic differentiation. Cells were fixed with 4% paraformaldehyde for 30 min. They were then stained with 0.4% Toluidine Blue in 0.2 M acetate buffer at pH 4.0.
Obtained samples were analyzed via Leica AF6000 microscope with a DFC 420C camera. To evaluate the effectiveness of adipogenic differentiation, we analyzed the percentage of cells bearing lipid droplets to the total number of cells in 12 independent fields of view for each experiment using the ImageJ software (NIH, Bethesda, MD, United States).
Alizarin Red was used to estimate the efficiency of osteogenic differentiation. Cells were fixed with 10% paraformaldehyde for 30 min. They were then stained using the Osteogenesis Quantitation Kit (Millipore) following the manufacturer’s instructions.
Toluidine blue staining was used to visualize chondrogenic differentiation. Cells were fixed with 4% paraformaldehyde for 30 min. They were then stained with 0.4% Toluidine Blue in 0.2 M acetate buffer at pH 4.0.
Obtained samples were analyzed via Leica AF6000 microscope with a DFC 420C camera. To evaluate the effectiveness of adipogenic differentiation, we analyzed the percentage of cells bearing lipid droplets to the total number of cells in 12 independent fields of view for each experiment using the ImageJ software (NIH, Bethesda, MD, United States).
10
Quantification of Calcium Deposition
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Osteogenesis quantitation kit (Millipore) was used for quantification of calcium depos-its. After alizarin red staining, 10% acetic acid was added to collect the cells and incubated at 85 °C for 10 min. Centrifuge the cell mixture at 16,000g for 20 min. Then, remove the supernatant and adjust pH value to 4.1–4.5 with 10% ammonium hydroxide. Read the absorbance at 405 nm on a spectrophotometer (Bio-rad).
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