Standard characterization of MSCs was performed by immunophenotyping of cell surface markers and differentiation into adipocytes, osteocytes, and chondrocytes. Immunophenotyping was performed using the Mouse Mesenchymal Stem Cell Marker Antibody Panel (R&D Systems). Flow cytometry analysis was performed using the FACSVantage™ system and CellQuest software. Differentiation of the MSCs into adipocytes, osteocytes, and chondrocytes was performed using the Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D Systems) [17 (link)].
Mouse mesenchymal stem cell functional identification kit
Manufactured by R&D Systems
Sourced in United States
The Mouse Mesenchymal Stem Cell Functional Identification Kit is a laboratory tool designed to identify and evaluate the functional characteristics of mouse mesenchymal stem cells (MSCs). The kit provides a set of reagents and protocols to assess the ability of mouse MSCs to differentiate into various cell lineages, such as osteoblasts, adipocytes, and chondrocytes.
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Lab products found in correlation
Mouse mesenchymal stem cell marker antibody panel, by R&D Systems (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
Cellquest software, by BD (1 mentions)
Cd105, by Abcam (1 mentions)
Facscalibur flow cytometer, by BD (1 mentions)
Canto 2, by BD (1 mentions)
Collagenase p, by Roche (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Glutamine, by Thermo Fisher Scientific (1 mentions)
Amphotericin b, by Thermo Fisher Scientific (1 mentions)
Dmem f 12 glutamax, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
α mem medium, by Merck Group (1 mentions)
Alcian blue 8gs, by Carl Roth (1 mentions)
Tissue tek oct compound, by Sakura Finetek (1 mentions)
Oil red o, by Thermo Fisher Scientific (1 mentions)
14 protocols using mouse mesenchymal stem cell functional identification kit
1
Characterization of Murine Mesenchymal Stem Cells
2
Chondrocyte Differentiation from Murine MSCs
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Chondrocyte differentiation was induced using Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D systems, Inc., Minneapolis, MN, USA), according to the manufacturer's instructions.
3
Osteogenic Differentiation of Mesenchymal Stem Cells
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Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D, Minneapolis, MN, USA) with osteogenic supplement was used according to the manufacturer instructions for culture during 28 days with culture medium replacement twice a week. After 28 days, OCs cultures were rinsed with distilled water (H2Od) and fixed with cold 70% ethanol for 1 h at room temperature (RT). Then, the ethanol was removed and cells were rinsed with H2Od. The calcium deposits were stained with Alizarin Red S (Sigma-Aldrich) (0.01 g/mL) for 1 h at RT and then the cultures were rinsed with H2Od to remove the excess stain.
4
Characterization of Expanded Renal Cells
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The characterization of ex vivo expanded cells from renal tissues was investigated by flow cytometry as previously described [14 (link)]. Briefly, twice-passaged cells were incubated with FITC or PE-conjugated antibodies against CD4, CD34, CD44, CD45, CD73, CD90, CD106, and MHC II (eBioscience), CD105 (Abcam), and FSP1 (Millipore), for 30 minutes. Isotype-identical antibodies served as negative control. Quantitative analysis was performed using a FACSCalibur flow cytometer with CellQuest software (Becton Dickinson). Differentiation of the twice-passaged cells into adipocytes, osteocytes, and chondrocytes was also examined by using the Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, USA), according to the manufacturer's instructions.
5
Phenotypic Characterization of ASCs
6
Mouse Adipose-Derived Stem Cell Isolation
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For isolation of mouse ADSC, inguinal adipose tissues were collected from 5- to 7-week-old C57BL/6 mice, followed by digestion with 0.1% collagenase P (#11213873001; Roche, Mannheim, Germany) in HBSS for 1 h at 37 °C with gentle shaking. The stromal vascular fraction (SVF) was then separated by centrifugation and treated with 1 × RBC lysis buffer to eliminate red blood cells. Cells from SVF were cultured in complete alpha-MEM. After overnight incubation, non-adherent cells were removed, and adherent cells were cultured until 80% confluency. The phenotype of the isolated ADSC was confirmed based on the positive expression of surface markers, including CD105, CD90, CD29, Sca-1, and CD44, and negative expression of CD11b, CD45, and CD34 (Supporting Information Fig. S1A–S11H ). Multipotency of the isolated ADSC was further demonstrated by adipocyte (Fig. S1I ), osteoblast (Fig. S1J ), and chondrocyte (Fig. S1K ) differentiation capacity using a Mouse Mesenchymal Stem Cell Functional Identification Kit (#SC010; R&D systems, Minneapolis, MN, USA).
Human ADSC (#STC002) were obtained from Stemore (Seoul, Republic of Korea). Mouse and human ADSC were routinely cultured in alpha-MEM containing 10% fetal bovine serum and 1% penicillin/streptomycin. Cells at passages between 3 and 5 were used for experiments.
Human ADSC (#STC002) were obtained from Stemore (Seoul, Republic of Korea). Mouse and human ADSC were routinely cultured in alpha-MEM containing 10% fetal bovine serum and 1% penicillin/streptomycin. Cells at passages between 3 and 5 were used for experiments.
7
Mesenchymal Stem Cell Lineage Differentiation
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F-SMSCs or M-SMSCs were cultured in StemXVivo mesenchymal stem cell expansion media (R&D Systems) and differentiation was induced as indicated using the media supplements included in the mouse mesenchymal stem cell functional identification kit (R&D Systems). Markers of osteocyte and chondrocyte lineages were detected using a sheep anti-mouse osteocalcin polyclonal antibody and a sheep anti-mouse collagen II antigen affinity-purified polyclonal antibody, respectively. In addition, the frozen sections were prepared to do the Oil Red O for lipid staining (Sigma-Aldrich).
8
Multilineage Differentiation Assay for BM-MSCs
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The Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Inc. Minneapolis, MN, USA) was used for multipotency evaluation of BM-MSC to differentiate osteoblasts and adipocytes. First, adipocytes were dyed with oil O red to demonstrate lipid droplets. Osteocytes were dyed with Von Kossa to demonstrate bone matrix deposits. BM-MSC culture without differentiation was used as the negative control. Adipocytes and macerated rat bone were positive controls for oil O red and Von Kossa, respectively.
9
Isolation and Characterization of Murine BM-MSCs
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We isolated a primary culture and propagated it in bone marrow-mesenchymal stem cells (BM-MSC) from mice. BM-MSC isolation and characterization were developed as described in our previous work [24 (link)]. Briefly: BM-MSC were obtained from 6 to 8-week-old Balb/c mice sacrificed in a CO2 chamber. Cells were deposited into a 25 cm2 culture flask (Corning Inc, One Riverfront Plaza, Corning, New York, NY 14831, USA) with DMEM/F12-GlutaMAX (Gibco, Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Life Technologies, Grand Island, NY, USA), gentamicin (100 µg/mL) (Gibco, Life Technologies, Grand Island, NY, USA), amphotericin B (2.5 µg/mL) (Gibco, Life Technologies, Grand Island, NY, USA), and glutamine (2 nm) (Gibco, Life Technologies, Grand Island, NY, USA). The following immunohistochemistry markers, CD105, CD90, and CD34, were used to identify MSC with the primary monoclonal antibodies anti-CD90, anti-CD105, and anti-CD34 (United States Biologicals, Salem, MA, USA), as the ISCT specifies [42 (link)]. In addition, the Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Inc. Minneapolis, MN, USA) was used for multipotency evaluation of BM-MSC to differentiate osteoblasts and adipocytes.
10
Isolation and Adipogenic Differentiation of BAMC
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BAMC were isolated and purified from 10–12 weeks-old WT and p53-null mice as described66 (link). Passaging was performed by replating the cells at 5 × 104 cells/cm2 in αMEM medium (Sigma M4526). BAMC differentiation into adipocytes was tested using an adipogenic supplement from the Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN; Cat.#SC010) according to the manufacturer’s instructions. The viability of cells was determined using ATP Cell Viability Assay, and CellTox Green Assay kits (Promega, Madison, WI).
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