Stempro osteogenesis kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

The StemPro® Osteogenesis Kit is a laboratory product designed for the induction and differentiation of stem cells into osteogenic (bone) cells. The kit provides a complete set of reagents, including culture medium and supplements, to support the osteogenic differentiation process. The core function of this product is to facilitate the controlled and efficient differentiation of stem cells into mature osteoblasts, which are the primary cell type responsible for bone formation.

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Lab products found in correlation

Stempro adipogenesis kit, by Thermo Fisher Scientific (4 mentions) Stempro chondrogenesis kit, by Thermo Fisher Scientific (3 mentions) Nunc 24 well plate, by Thermo Fisher Scientific (2 mentions) Alizarin red, by Merck Group (1 mentions) Toluidine blue, by Merck Group (1 mentions) Celltracker green cmfda, by Thermo Fisher Scientific (1 mentions) Chondrogenesis differentiation kit, by Thermo Fisher Scientific (1 mentions) Oil red o, by Merck Group (1 mentions) Celigo imaging cytometer, by Revvity (1 mentions) Phosphatase substrate, by Merck Group (1 mentions) Multiskan ex elisa plate reader, by Thermo Fisher Scientific (1 mentions) 12 well plate, by Corning (1 mentions) Formaldehyde, by Merck Group (1 mentions) Phosphate buffered saline (pbs), by Lonza (1 mentions)

Close spelling variants of this product

StemPro Osteogenesis Diff Kit StemPro adipogenesis and osteogenesis differentiation kit StemPro Osteogenesis Differentiation Kit StemPro Osteogenesis Differentiation Media Kit Stempro® Osteogenesis Differentiation kit medium StemPro Osteogenesis StemPro kits Osteogenesis

8 protocols using stempro osteogenesis kit

Osteogenesis Quantification by Alizarin-Red

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The P4 cells were plated at a density of 5 × 10⁴ in a 24 well plate. The cells were cultured in CCM until they reached 80% confluence. Upon confluence, we replaced the regular CCM with osteogenic media (StemPro osteogenesis kit, ThermoFisher Scientific, Waltham, MA, USA) with freshly prepared LPS solution at 5, 10, and 20 µg/mL concentrations. The osteogenic media with LPS was replaced every fourth day for a total of 28 days.
Alizarin-Red staining: At the end of Day 28, the cells were fixed using 4% formaldehyde. The fixed cells were washed in PBS to stain further using Alizarin-Red (MERCK, Darmstadt, Germany). The stained cells’ photomicrographs were captured using a 12-megapixel digital camera attached to a Nikon eclipse Ti-U inverted bright field microscope.
Extraction of Alizarin-Red stain for osteogenic quantification—the cetylpyridinium chloride (CPC) (MERCK, Darmstadt, Germany) extraction method was employed to extract the stain. Briefly, 350 mg CPC was dissolved in 10 mL of double-distilled water. We added an appropriate amount (2.3 mL) of the prepared CPC solution into each well, and the plate was incubated at 37 °C for 2 h with mild shaking. The plate was read using a microplate reader (infinite 200) at 405 nm wavelength. The absorbance readings were tabulated for statistical analysis.

Banavar S.R., Rawal S.Y., Pulikkotil S.J., Daood U., Paterson I.C., Davamani F.A., Kajiya M., Kurihara H., Khoo S.P, & Tan E.L. (2021). 3D Clumps/Extracellular Matrix Complexes of Periodontal Ligament Stem Cells Ameliorate the Attenuating Effects of LPS on Proliferation and Osteogenic Potential. Journal of Personalized Medicine, 11(6), 528.

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Osteogenic Differentiation of C-MSCs

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The clumps of cells were treated with osteoinductive medium (StemPro osteogenesis kit) (ThermoFisher, Waltham, MA, USA) with 5, 10, or 20 µgm/mL LPS for four weeks. C-MSCs treated with osteoinductive media (OIM) alone formed the positive control group, while C-MSCs treated with regular CCM formed the negative control group.

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Mesenchymal Stem Cell Lineage Differentiation

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For osteogenic differentiation, AD-MSCs were seeded at a density of 1.6 × 10⁴ cells/cm² in Nunc™ 24-well plates (Thermo Fisher Scientific) or at a density of 1 × 10⁴ in 96-well plates (TPP). For adipogenic differentiation, cells were cultured at a density of 1.6 × 10⁴ cells/cm² in Nunc™ 24-well plates (Thermo Fisher Scientific). Differentiation was started 24 h after seeding with StemPro® Osteogenesis Kit or StemPro® Adipogenesis Kit (Gibco/Life Technologies). For chondrogenic differentiation, cells were cultured at a density of 5 × 10³ cells/cm² in a Nunc™ 24-well plate (Thermo Fisher Scientific) and differentiation was induced at the 4th day of culture using the StemPro® Chondrogenesis Kit (Gibco/Life Technologies). All media were changed every 4 days.

Canepa D.D., Casanova E.A., Arvaniti E., Tosevski V., Märsmann S., Eggerschwiler B., Halvachizadeh S., Buschmann J., Barth A.A., Plock J.A., Claassen M., Pape H.C, & Cinelli P. (2021). Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry. Stem Cell Research & Therapy, 12, 7.

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Multilineage Differentiation of Bone Marrow Cells

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Cells were sorted directly into culture at a 96-well plate. The cultures were incubated at 37°C in a humidified atmosphere with 5% O₂ and 10% CO₂ for 7–10 days. Colonies were stained by CellTracker™ Green CMFDA (Life Technologies), and image was acquired by Celigo Imaging Cytometer (Nexcelom). For in vitro differentiation, clonally expanded Ncad-CreER^T driven Tomato⁺ BM and bone stromal cells were isolated from CFU-F cultures by digesting with 0.25% Trypsin/EDTA, split into 3 aliquots, and seeded into separate cultures permissive for differentiation: StemPro Osteogenesis kit GIBCO A10072–01, Adipogenesis kit A10070–01 and Chondrogenesis differentiation kit A10071–01. The osteoblastic differentiation was assessed by VECTOR Red Alkaline Phosphatase; the adipogenic differentiation was detected by Oil Red O (Sigma); and the chondrogenic differentiation was detected by Toluidine blue (Sigma, 0.1g T Blue/100mL distilled water).

Zhao M., Tao F., Venkatraman A., Li Z., Smith S.E., Unruh J., Chen S., Ward C., Qian P., Perry J.M., Marshall H., Wang J., He X.C, & Li L. (2019). N-Cadherin-Expressing Bone and Marrow Stromal Progenitor Cells Maintain Reserve Hematopoietic Stem Cells. Cell reports, 26(3), 652-669.e6.

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Multilineage Characterization of Adipose-Derived Stem Cells

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Adipose-derived stem cells were characterized by flow cytometry and multilineage differentiation. For flow cytometry, ASCs in suspension were incubated with phycoerythrin-coupled antibodies for rat CD31 (BD Pharmingen, San Jose, CA), CD44 (eBioscience Inc, San Diego, CA), CD45 (BioLegend, San Diego, CA), and CD90 (Acris Antibodies, San Diego, CA) in the dark, at room temperature for 30 minutes. The cells were washed with wash buffer (0.5% fetal bovine serum in phosphate-buffered saline) and were fixed in neutral 4% paraformaldehyde solution for 30 minutes.
To induce adipogenic and osteogenic differentiation, ASCs were cultured in adipogenic differentiation medium (StemPro Adipogenesis Kit, Gibco) and osteogenic differentiation medium (StemPro Osteogenesis Kit, Gibco) for 14 and 21 days, respectively. For chondrogenic differentiation, ASCs micromass pellets were fed with chondrogenic differentiation medium that consisted of Dulbecco modified eagle medium with 10-ng/mL transforming GF β-3 [TGFβ-3], and 200-μmol/L ascorbic acid for 2 weeks. Adipogenic, osteogenic, and chondrogenic differentiation was confirmed with oil red O, alizarin red staining, and alcian blue staining, respectively.

Orbay H., Little C.J., Lankford L., Olson C.A, & Sahar D.E. (2015). The key components of Schwann cell-like differentiation medium and their effects on gene expression pattern of adipose-derived stem cells. Annals of plastic surgery, 74(5).

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Multi-lineage Differentiation Potential

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The multi‐lineage potential of the cells was ascertained by inducing the samples post‐harvest with the StemPro Adipogenesis kit, StemPro Chondrogenesis kit and StemPro Osteogenesis kit (Life Technologies, UK). The media were prepared and used as per the manufacturer's instructions and the differentiation studies were performed as described in 30.

Rafiq Q.A., Coopman K., Nienow A.W, & Hewitt C.J. (2016). Systematic microcarrier screening and agitated culture conditions improves human mesenchymal stem cell yield in bioreactors. Biotechnology Journal, 11(4), 473-486.

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Osteogenic Differentiation Assay

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Cells were seeded in 12-well plates (Corning) (10⁴ cells/cm²) in triplicate. After 3 days, the medium was replaced with an osteogenic induction medium (StemPro Osteogenesis Kit, Life Technologies); in parallel, negative controls were cultivated in MSC medium. Differentiation and the MSC media were changed every 2–3 days. After 9 days, alkaline phosphatase (ALP) activity was quantified through incubation with phosphatase substrate (Sigma-Aldrich), and the resulting p-nitrophenol was quantified colourimetrically using Multiskan EX ELISA Plate Reader (Thermo Scientific) at 405 nm. Absorbance data were normalised by subtracting from undifferentiated, negative controls.

Romanelli Tavares V.L., Guimarães-Ramos S.L., Zhou Y., Masotti C., Ezquina S., Moreira D.D., Buermans H., Freitas R.S., Den Dunnen J.T., Twigg S.R, & Passos-Bueno M.R. (2021). New locus underlying auriculocondylar syndrome (ARCND): 430 kb duplication involving TWIST1 regulatory elements. Journal of Medical Genetics, 59(9), 895-905.

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Comprehensive Characterization of hMSCs

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Multi‐parameter immunophenotypic analysis of the hMSCs was determined by flow cytometry and the antibodies were selected on the basis of the panel recommended by the International Society for Cellular Therapy (ISCT) (Dominici et al., 2006). This was performed using a Guava HT flow cytometer with excitation at 488 nM and is described in detail in Chan et al. (2014). Tri‐lineage differentiation potential of the cells was determined by using the StemPro Adipogenesis kit, StemPro Chondrogenesis kit and StemPro Osteogenesis kit (Life Technologies, ThermoFisher Scientific Company, Loughborough, UK) in accordance with the manufacturer's instructions on post‐harvest hMSC samples to induce differentiation. Staining of the samples to determine differentiation was performed as described in (Rafiq et al., 2013b). CFU‐f efficiency was determined by culturing the hMSCs in monolayer at a cell density of 10 cells/cm² and with a complete medium exchange every 4 days. After 15 days in culture, hMSCs were washed with PBS (Lonza, Visp, Switzerland) and fixed with 4% formaldehyde (v/v) (Sigma, UK) for 30 min. A 1% crystal violet (Sigma–Aldrich) in 100% methanol (w/v) was used for colony staining. The samples were incubated at room temperature for 30 min in the 1% crystal violet solution. All stained colonies that comprised of more than 25 cells were recorded as CFUs.

Rafiq Q.A., Hanga M.P., Heathman T.R., Coopman K., Nienow A.W., Williams D.J, & Hewitt C.J. (2017). Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor. Biotechnology and Bioengineering, 114(10), 2253-2266.

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