All MSCs were individually differentiated in adipogenic (Cat No. GUXMX-90031, Cyagen Biosciences, Santa Clara, CA, USA) and osteogenic differentiation medium (Cat No. GUXMX-90021, Cyagen Biosciences). The cells in the adipogenic and osteogenic differentiation medium were then stained with oil red O (Sigma-Aldrich, St. Louis, MO, USA) and von Kossa (Sigma-Aldrich) for 10 minutes. For the quantification of mineralization by measuring calcium nodules, the differentiated cells were stained with alizarin red S and resolved with acetic acid. The supernatant was analyzed by measuring the absorbance at 490 nm using a spectrophotometer. The differentiation steps used for both adipocytes and osteoblasts were determined from established protocols [6 (link),17 ].
Von kossa
Manufactured by Merck Group
Sourced in United States
The Von Kossa stain is a histochemical technique used to identify the presence of calcium deposits in biological samples. It is a widely used method in histology and pathology laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Oil red o, by Merck Group (4 mentions)
Alizarin red s, by Merck Group (2 mentions)
Guxmx 90021, by Cyagen (1 mentions)
Guxmx 90031, by Cyagen (1 mentions)
Alizarin red, by Merck Group (1 mentions)
Calcium colorimetric assay kit, by Merck Group (1 mentions)
Dmem lg, by Thermo Fisher Scientific (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
β glycerol phosphate, by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Matrigel plug, by Corning (1 mentions)
Quant it picogreen dsdna assay, by Thermo Fisher Scientific (1 mentions)
Safranin o, by Merck Group (1 mentions)
Sudan 3, by Merck Group (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Toluidine blue stain, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Ab34712, by Abcam (1 mentions)
Dulbecco modified eagle medium (dmem), by Capricorn (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
10 protocols using von kossa
1
Adipogenic and Osteogenic Differentiation Assay
2
Osteoid Identification in N. furzeri
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Formaldehyde-fixated and methyl methacrylate-embedded N. furzeri were stained with von Kossa/toluidine blue. This staining was performed to verify the presence of osteoid in the vertebral area of N. furzeri as it allows to differentiate between mineralized bone and unmineralized osteoid. The same toluidine blue solution, as described above, was used. Additionally, von Kossa staining (Sigma-Aldrich) was performed to demonstrate the presence of calcium deposits.
3
Quantifying Aortic Calcium Deposition
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Calcium mineral deposits in aortic sections were detected by Alizarin Red and Von Kossa staining (Sigma, St. Louis, MO) as described previously6 (link). The percentage of positively stained area for each aortic section was quantified using ImageJ software (NIH). Calcium content in the aortic tissues was measured as previously described6 (link). Dissected aortas were briefly incubated at 70 °C for 30 min to obtain their dry weight and decalcified with 0.6 mM HCl at 37 °C for 48 h. The vascular calcium concentration was quantified using the Calcium Colorimetric Assay Kit (Sigma, St. Louis, MO) and normalized by the dry weight of the aortas, which was expressed as fold-change compared with control.
4
Osteogenic Differentiation of NBM-MSCs and DBM-MSCs
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NBM-MSCs and DBM-MSCs (1×104 cells/cm2) were seeded in 6-well plates in DMEM-low glucose (DMEM-LG; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS, 0.1 μM dexamethasone, 10 mM β-glycerol phosphate and 50 μM ascorbic acid (all Sigma-Aldrich, St. Louis, MO, USA), and the medium was replaced every 3 days. Cell morphology was observed under an inverted fluorescence microscope (ECLIPSE TE2000-S). Von Kossa (Sigma-Aldrich) staining was used to reveal mineralized areas.
5
Mesodermal Differentiation Potential of PD-MSCs
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To confirm the mesodermal differentiation potential, PD-MSCs PEDF (passage = 5) were seeded in each plate (5 × 10 3 cells/cm 2 ) with each differentiation induction medium for ~21 days using the StemPro Adipogenesis and Osteogenesis Differentiation Kit (Invitrogen) according to the manufacturer's instructions. After 21 days, the cells were fixed in 4% paraformaldehyde and stained using oil red O (Sigma-Aldrich) for lipid droplet identification, and von Kossa (Sigma-Aldrich) staining with 5% silver nitrate was used to visualize calcium deposits. Each experiment was performed in triplicate.
6
Matrigel Implantation of Progenitor Cells
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Eight–ten-week-old WT male mice were anesthetized and shaved on the left flank and abdomen before sterilization of the surgical site. Recipients for these experiments were syngeneic with donors. A 5-µL Matrigel plug (Corning, 356231) containing 8,000 to 10,000 sorted Ctsk-lineage PSCs, PP1, or PP2 cells was implanted underneath the renal capsule. Animals were killed by CO2 after 3 wk. Kidneys were fixed with 4% PFA for 5 h and subjected to infiltration, embedding, and sectioning. Von Kossa (Sigma-Aldrich) staining was performed to detect mineralized nodule formation. Quantification of Von Kossa staining was performed using Image J (NIH.gov, version 1.53t).
7
Osteogenic Differentiation of MSCs and ASCs
8
Multilineage Differentiation Assays of MSCs
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Specific differentiation media were used for the osteogenic, chondrogenic, and adipogenic induction of undifferentiated MSC cultures in vitro as reported previously [12 (link)–14 (link)]. Safranin O (Sigma), Oil Red O (Sigma), Sudan III (Sigma), and von Kossa (Sigma) stainings were performed for the determination of the outcome of the differentiation assays.
9
Multilineage Differentiation of hUC Fibroblasts
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In-vitro differentiation assay was performed on culture-expanded hUC fibroblasts between passages 1–3. The cells were passaged in MSC proliferating medium for 1–2 days followed by incubating with complete Adipogenic, Osteogenic, and Chondrocyte differentiation medium as per manufacturers protocol (HiMedia Laboratories Pvt. Ltd., India) to induce adipogenesis, osteogenesis, and chondrogenesis. The stimulation for cellular differentiation was carried out for 2–4 weeks, and the media was replaced with fresh media after every three days. After 2–4 weeks of incubation, the spent medium was removed, and ice-cold methanol was added to the cells and kept at − 20 °C for 10 min. The cells were then stained with 0.3% Oil Red O, Von Kossa, and 0.1% Toluidine blue stain, pH 2.0–2.5 (Sigma-Aldrich, St. Louis, MO, USA) as per manufacturers' recommendation. The experiment was performed in triplicates, the cells were observed under an inverted microscope (Olympus, Tokyo, Japan), and images for trilineage differentiation were captured using Leica Optica Image Viewer software.
10
Chitosan Microparticles for Bone Regeneration
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Chitosan with medium molecular weight powder was purchased from Sigma-Aldrich and used for preparation of composites. Sparus aurata fishes were obtained from a commercial dealer in Izmir. Filter (Partec CellTrics, Fisher Scientific) was used with 100 μm mesh diameters to obtain FS microparticle. Tris hydrochloride (Tris-HCl), ethylenediaminetetraacetic acid (EDTA) and sodium dodecyl sulfate (SDS) were used for decellularization process and supplied from Applichem. WST-1 cell proliferation reagent (BioVision Inc.) and StemTAG ALP Assay kit (Cell Biolabs Inc.), Enzyline PAL Optimise, ALP Kit (Biomeriux Inc.), Human Osteocalcin (OC/BGP) ELISA Kit (Elabscience) were used for in vitro assays. DAPI (Sigma Aldrich) and Alexa Fluor 488 (Thermo Fisher Scientific) were used for fluorescence staining. Materials were used for histological stainings were Hematoxylin&eosin (Surgipath), Masson Trichrome (GBL, Turkey), Type I collagen antibody (Bioss, bs0578-R, Pro-Collagen I Polyclonal Antibody) and type II collagen antibody (Abcam, ab34712, Rabbit Polyclonal Collagen II Antibody). Alizarin Red S and von Kossa were purchase from Sigma, Aldrich. DMEM (Capricorn Scientific) and all supplements (Lonza) were used for cell culture study.
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