We purchased Whatman 114 filter paper, glycerol-2-phosphate, ascorbic acid, alizarin red S and 4,6-diamidino-2-phenylindole (DAPI) from Sigma-Aldrich (St Louis, MO). We obtained the paraformaldehyde solution (16% (v/v)) from Electron Microscopy Sciences (Hatfield, PA). Mouse antibodies to Osteocalcin and Alexa 647-labeled antibodies to rabbit IgG were supplied by Abcam (Cambridge, MA). We purchased trypsin-EDTA, penicillin-streptomycin, phalloidin (Texas Red-X), fetal calf serum (FCS), fetal bovine serum (FBS), alpha-minimal essential medium (α-MEM) medium, and Dulbecco’s phosphate buffered saline (DPBS) from Invitrogen (Carlsbad, CA). We bought the OsteoImage kit from Lonza Walkersville Inc. (Walkersville, MD). We used all of the reagents as received without further purification.
Osteoimage kit
Manufactured by Lonza
Sourced in Switzerland, Germany, United States
The OsteoImage kit is a laboratory equipment product designed for the detection and quantification of osteogenic cells. It provides a direct and reliable method for measuring osteogenic activity in cell culture systems. The kit includes specialized reagents and protocols for the assay.
Automatically generated - may contain errors
Lab products found in correlation
Alizarin red s, by Merck Group (2 mentions)
Ascorbic acid, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Paraformaldehyde solution, by Electron Microscopy Sciences (1 mentions)
Osteocalcin, by Abcam (1 mentions)
Glycerol 2 phosphate, by Merck Group (1 mentions)
Whatman, by Cytiva (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Eclipse ti inverted microscope, by Nikon (1 mentions)
Fv1200, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Oil red o, by Olympus (1 mentions)
Oil red o, by Merck Group (1 mentions)
Model ckx41, by Olympus (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
7 protocols using osteoimage kit
1
Osteogenic Differentiation Assay
2
Quantifying Osteogenic Differentiation in ADSCs
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At Day 14 or 21 of culture, cells cultured in 24-well plates were fixed in 10% buffered formalin (Sigma Aldrich). The accumulated bone mineral was fluorescently stained with OsteoImage kit (Lonza Ltd., Basel, Switzerland) according to the manufacturer’s instructions. Cell nuclei were counterstained with Hoechst 33,342 (Sigma Aldrich). Undifferentiated ADSCs were stained following the same procedure to validate bone mineral staining specificity. The bone mineral was semiquantified by measuring fluorescence intensity at 488/520 nm, while the relative number of cells was semiquantified by measuring Hoechst 33,342 florescence intensity at 355/460 nm (excitation/emission). Fluorescence intensity was measured in 10 points evenly distributed within a well. The relative quantity of bone mineral was expressed as OsteoImage fluorescence intensity normalized to Hoechst 33,342 fluorescence intensity. Each sample was assayed in quadruplicate. Additionally, stained samples were observed under a Nikon Eclipse-Ti inverted microscope (Nikon Instruments Inc., Melville, NY, USA) for image acquisition.
3
Microscopy Analysis of Bone and Lipid Deposition
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For microscopy analysis, cultures were fixed in 4% paraformaldehyde (PFA; Sigma-Aldrich) for 30 min at room temperature. To characterise bone-like calcium deposits in monolayers, cultures were stained with Alizarin Red S (Sigma-Aldrich). To identify hydroxyapatite deposits, monolayers and microtissues were stained using the OsteoImage kit (Lonza) as per the manufacturer’s instructions. To identify lipid vacuoles, monolayers and microtissues were stained with Oil Red O (Sigma-Aldrich). Where indicated, nuclei were stained with 4′,6-Diamidino-2-Phenylindole (DAPI; ThermoFisher). Monolayer and microtissue bright field microscope images were captured using an Olympus microscope (Model CKX41 equipped with X-Cite 120Q Fluorescence Illuminator). OsteoImage and Oil Red O 3D microtissue images were captured using a confocal microscope (FV1200, Olympus).
4
Quantifying Osteogenic Mineralization in MC3T3-E1 Cells
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Mineralization of MC3T3-E1 cells was assessed by the OsteoImage kit (Lonza, Basel, Switzerland) based on fluorescent staining of hydroxyapatite in cell deposits. Briefly, cells cultured at different time points were fixed, stained with the fluorescent reagent, and measured at excitation wavelength 492 nm and emitted wavelength 520 nm and observed using a fluorescence microscope (Olympus, Shinjuku City, Tokyo, Japan).
5
Murine Osteoblast Differentiation in 3D Hydrogels
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Osteoblast (here murine MC3T3-E1) differentiation induced by the hydrogel constructs was studied based on matrix maturation and mineralization. Alkaline phosphatase (ALP) is an osteoblastic differentiation marker that is expressed in the initial phases of the differentiation process. The deposition of HA into the extracellular matrix was visualized by the Osteoimage kit (Lonza, Germany). In this test, a cell density of 1 × 10 5 cells per well was added to the 3D printed constructs, and after reaching 100% confluency (in 7 days), the osteogenic medium, including the culture medium plus 10 mmol/L β-glycerophosphate, 0.05 mmol/l ascorbic acid, and 10 nmol/l dexamethasone (all from Sigma), was replaced with the previous medium, i.e., α-MEM.
Afterwards, the osteogenic medium was added freshly every 72 h.
Afterwards, the osteogenic medium was added freshly every 72 h.
6
Osteogenic Differentiation of hASCs on SiOC
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The hASCs were seeded on the SiOC scaffolds and cultivated for 7 days. Then, the culture medium was changed to Mesenchymal Stem Cell Osteogenic Differentiation Medium (Lonza), with medium changes every 2–3 days. After 21 days of treatment with an induction medium, the scaffolds were fixed for fluorescence and scanning electron microscopy analysis. To identify hydroxyapatite deposition, the scaffolds were incubated with the OsteoImage Kit (Lonza, PA-1503) according to the manufacturer’s instructions and analyzed under DMI6000B fluorescence microscope (Leica). Three technical replicates were performed.
7
Osteogenic Differentiation of HUCPV on WE43-PEO
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HUCPV were isolated with the approval from the local ethical committee “Ethik-Kommission der Ärztekammer Hamburg” (Hamburg, Germany). The written consent of the donor was obtained. The isolation and preparation of the cells was achieved as shown by Cecchinato et al. [30 (link)]. After cutting the umbilical cord into 5 cm pieces, they were cultured for 10 days in T-175 cell culture flask with α-MEM, 15% FBS and 1% antibiotics. When cells were outgrowing, the medium was changed every 2–3 days. When cells recached 80% confluency, they were detached and transferred into fresh medium.
100,000 human umbilical cord perivascular cells (HUCPV) were seeded onto four WE43-PEO variants with phosphate-based surface-coating under cell culture conditions: PEO5, PEO8, PEO9 and PEO10, and allowed to attach overnight. On the next day, the medium was changed to osteogenesis medium. After 3 weeks of osteogenic differentiation, the WE43 variants were stained with live-dead markers (Invitrogen, Carlsbad, CA, USA, catalogue: L-3224) and an OsteoImage kit (Lonza, Walkersville, MD, USA, catalogue: PA-1503) according the instructions of the manufacturer. The images were visualized using a fluorescence microscope (Nikon ECLIPSE Ti-S/L100, Nikon GmbH, Düsseldorf, Germany) and qualitatively analyzed.
100,000 human umbilical cord perivascular cells (HUCPV) were seeded onto four WE43-PEO variants with phosphate-based surface-coating under cell culture conditions: PEO5, PEO8, PEO9 and PEO10, and allowed to attach overnight. On the next day, the medium was changed to osteogenesis medium. After 3 weeks of osteogenic differentiation, the WE43 variants were stained with live-dead markers (Invitrogen, Carlsbad, CA, USA, catalogue: L-3224) and an OsteoImage kit (Lonza, Walkersville, MD, USA, catalogue: PA-1503) according the instructions of the manufacturer. The images were visualized using a fluorescence microscope (Nikon ECLIPSE Ti-S/L100, Nikon GmbH, Düsseldorf, Germany) and qualitatively analyzed.
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