The multilineage differentiation potential of hPVCs cultured in NG and HG conditions was evaluated at passages 3 and 4 as previously described [13 (link)]. Briefly, for adipogenic differentiation, hPVCs (4 × 104 cells/well) were plated in 12-well tissue culture plates and cultured in adipogensis differentiation medium (A10070-01; StemPro®, Thermo Fisher Scientific, Waltham, MA, USA) for 21 days. After 21 days, the cultures were stained with Oil Red O (CM-005; LifeLine Cell Technology, CA, USA) to visualize the intracellular accumulation of lipid vacuoles. For osteogenic differentiation, hPVCs (4 × 104 cells/well) were seeded in 12-well tissue culture plates and cultured in osteogenesis differentiation medium (A100-01; StemPro®, Thermo Fisher Scientific, Waltham, MA, USA) for 21 days. On day 21, the cells were stained with Alizarin Red S (Lifeline Cell Technology) to visualize the mineralization in osteogenic cell cultures.
A10070 01
Manufactured by Thermo Fisher Scientific
Sourced in United States
The A10070-01 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for general laboratory use. The core function of this product is to provide a specific technical capability for laboratory applications, but detailed specifications are not available in this unbiased, factual description.
Automatically generated - may contain errors
Lab products found in correlation
A1007201, by Thermo Fisher Scientific (14 mentions)
A1007101, by Thermo Fisher Scientific (11 mentions)
Alcian blue, by Merck Group (4 mentions)
Oil red o, by Merck Group (4 mentions)
Alizarin red s, by Merck Group (3 mentions)
Alizarin red s, by Lifeline Cell Technology (1 mentions)
Stempro, by Thermo Fisher Scientific (1 mentions)
Dxflex flow cytometry system, by Beckman Coulter (1 mentions)
Ab8158, by Abcam (1 mentions)
Ab23830, by Abcam (1 mentions)
Facscanto 2, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
Software, by FlowJo (1 mentions)
Facscalibur system, by BD (1 mentions)
Facscelesta flow cytometer, by BD (1 mentions)
Cd105, by BD (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Trypan blue, by Thermo Fisher Scientific (1 mentions)
Cd105, by Bioss Antibodies (1 mentions)
Accuri c6 plus flow cytometer, by BD (1 mentions)
19 protocols using a10070 01
1
Multilineage Differentiation of hPVCs
2
Multilineage Differentiation of MSCs
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To confirm MSC identify, CD43−CD45− monolayer cells were sorted at various days of culture and differentiated per manufacturer’s instructions into adipocytes (A1007001, Thermo Fisher Scientific), chondrocytes (A1007101, Thermo Fisher Scientific), and osteocytes (A1007201, Thermo Fisher Scientific). For comparison, control MSCs derived from the BM of a healthy individual were differentiated following the same procedures.
3
Triamcinolone Impacts BM-MSC Characteristics
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Impact of TP at 10-8 M concentration on the characteristic features of BM-MSCs were determined by investigating cell surface markers and by evaluating BM-MSCs’ differentiation capacities. For immunophenotyping, 2x105 BM-MSCs were seeded into 6-well tissue culture plates as triplicates, incubated in the presence of 10-8 M TP for 24 hours followed by collecting cells with trypsinization and labeling with antibodies abovementioned. Cells were immediately read with Beckman Coulter DxFLEX flow cytometry system and analysis was performed on CytExpert software. For evaluation of BM-MSCs’ differentiation capacities, 1x104 BM-MSCs were seeded into 96-well cell culture plates and once they reached 80% confluency, differentiation was initiated by commercial chondrogenesis (Thermo Fisher Scientific, #A1007101), osteogenesis (Thermo Fisher Scientific #A1007201), and adipogenesis (Thermo Fisher Scientific, #A1007001) kits, either supplemented with 10-8 M TP or not. Differentiation media were replaced twice a week. On the 21st day of differentiation, cells were fixed with 10% neutral-buffered formalin solution. Chondrogenesis, osteogenesis, and adipogenesis were evaluated by Alcian Blue, Alizarin Red, and Oil Red-O staining, respectively.
4
Multilineage Differentiation of Murine MSCs
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Murine bone marrow mesenchymal cells (105 cells/well, 24-well plate) were incubated in an osteogenic medium (A1007201; Thermo Fisher Scientific Inc., Waltham, MA, USA) for 18 days. Mineralized extracellular matrices were probed using von Kossa staining [26 (link)]. For adipocyte formation, 105 cells were incubated in adipogenic medium (A1007001; Thermo Fisher Scientific Inc., Waltham, MA, USA) for 15 days [22 (link)]. Adipocytes were probed using Nile red staining kit. In some experiments, a total of 105 cells/well (24-well plates) were incubated in a differentiation medium for brown adipocytes (BADTM, Cosmo Bio Co., Ltd., Tokyo, Japan) and white adipocytes (WATDM, Cosmo Bio Co., Ltd., Tokyo, Japan) for 15 days, respectively, according to the maker’s instructions.
5
Multilineage Differentiation of MSCs
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Differentiation experiments were carried out in technical triplicates and maintained in culture for three weeks. The media were exchanged every three days. The controls were cultured in DMEM supplemented with 10% FCS, 1% Pen/Strep and 1% amphotericin B. For adipogenic and osteogenic differentiation, 4000 MSCs were seeded per well of a 12-well plate in DMEM supplemented with 10% FCS, 1% Pen/Strep and 1% amphotericin B for 48 h. After washing with PBS +/+, adipogenesis (ThermoFisher, Waltham, MA, USA, A1007001) or osteogenesis (ThermoFisher, A1007201) differentiation media were added. For chondrogenic differentiation, 350,000 MSCs were pelleted per 15 mL Falcon tube and resuspended in chondrogenesis differentiation media (ThermoFisher, A1007101).
6
Multilineage Differentiation of MSCs
7
Adipogenic Differentiation of hMSCs
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Human mesenchymal stem cells were kindly given by Prof. Chan Hon Fai Vivas (School of Biomedical Sciences, The Chinese University of Hong Kong) and differentiated into adipocytes following the manufacturer’s instructions (Thermo Fisher, A1007001). The differentiation medium was changed every 2–3 days.
8
Multilineage Differentiation of Mesenchymal Stem Cells
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Experimental differentiation procedures were conducted in triplicates (passage 2), and the cultures were maintained for a duration of three weeks. The cultural media were refreshed every three days. The control samples were cultured in DMEM supplemented with 10% FCS, 1% Pen/Strep, and 1% amphotericin B. Adipogenic and osteogenic differentiations were performed by seeding 4000 MSCs per well of a 12-well plate in DMEM supplemented with 10% FCS, 1% Pen/Strep, and 1% amphotericin B for 48 h. Subsequently, the cells were washed with PBS and treated with either adipogenesis ((ThermoFisher, Waltham, MA, USA, A1007001)) or osteogenesis (ThermoFisher, A1007201) differentiation media. For chondrogenic differentiation, 350,000 MSCs were collected in 15 mL Falcon tubes and pelleted, followed by resuspension in chondrogenesis (ThermoFisher, A1007101) differentiation media.
9
Isolation and Characterization of Murine MSCs
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Bone marrow MSCs were harvested from the femurs, tibia, and humerus of BALB/c mice following a methodology described by Amend et al. [19 (link)] and cultivated for three days. The medium was refreshed at intervals of three to four days until adherent cells reached 90% confluence, being defined at this stage as MSCs at passage zero (P0).
For MSC characterization, the capacity of these cells to differentiate into adipocytes and osteoblasts in vitro was evaluated using specific differentiation medium for the assessment of adipogenesis or osteogenesis (# A1007001; #A1007201, Gibco Invitrogen, USA), assessment of the expression of the common markers CD73, CD45 (#sc-14682; #sc-25590, Santa Cruz Biotechnology, USA) and CD34 (#ab8158, Abcam, UK) by immunofluorescence and analysis of the expression of CD11b, CD45 (#11-0112-82, #48-0451-82 eBioscience), CD34 (#ab23830 –Abcam, UK), CD73 and CD105 (#127209; #120407—BioLegend, Inc. USA) by flow cytometry (FACSCanto™ II (BD Biosciences) using FACSDiva software (BD Biosciences). The flow cytometry data were analyzed using FlowJo software (FlowJo, OR, USA).
For MSC characterization, the capacity of these cells to differentiate into adipocytes and osteoblasts in vitro was evaluated using specific differentiation medium for the assessment of adipogenesis or osteogenesis (# A1007001; #A1007201, Gibco Invitrogen, USA), assessment of the expression of the common markers CD73, CD45 (#sc-14682; #sc-25590, Santa Cruz Biotechnology, USA) and CD34 (#ab8158, Abcam, UK) by immunofluorescence and analysis of the expression of CD11b, CD45 (#11-0112-82, #48-0451-82 eBioscience), CD34 (#ab23830 –Abcam, UK), CD73 and CD105 (#127209; #120407—BioLegend, Inc. USA) by flow cytometry (FACSCanto™ II (BD Biosciences) using FACSDiva software (BD Biosciences). The flow cytometry data were analyzed using FlowJo software (FlowJo, OR, USA).
10
Effect of FIR on TMSC Differentiation
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After exposure of TMSC to FIR irradiation for 0 or 30 min, cells were collected and stained with phycoerythrin (PE)-conjugated anti-human CD14, CD34, CD73, and fluorescein isothiocyanate (FITC)conjugated anti-human CD45, CD90, CD105 antibodies. Stained cells were analyzed using a FACSCalibur system (BD Biosciences, San Diego, CA, USA). All antibodies were purchased from BD Biosciences.
Adipogenic or osteogenic differentiation and drug treatments TMSC at a confluence of 80-90% were exposed to FIR irradiation for the indicated times and incubated in commercially available adipogenic or osteogenic differentiation medium (Cat. No. A10070-01 or A10072-01; Invitrogen) for up to 14 days. The culture medium was changed every 3 or 4 days. In some experiments, TMSC were pretreated with CsA (0, 0.25, 0.5, and 1 μM; Sigma-Aldrich) before exposure to FIR irradiation and then subjected to differentiation. After differentiation, cells were fixed with 4% formalin for 30 min and washed with phosphate-buffered saline. Adipogenic differentiated cells were stained with Oil Red O solution and osteogenic differentiated cells with Alizarin red S solution for 1 h at room temperature. The remaining excessive staining solution was removed, and stained cells were visualized under a microscope.
Adipogenic or osteogenic differentiation and drug treatments TMSC at a confluence of 80-90% were exposed to FIR irradiation for the indicated times and incubated in commercially available adipogenic or osteogenic differentiation medium (Cat. No. A10070-01 or A10072-01; Invitrogen) for up to 14 days. The culture medium was changed every 3 or 4 days. In some experiments, TMSC were pretreated with CsA (0, 0.25, 0.5, and 1 μM; Sigma-Aldrich) before exposure to FIR irradiation and then subjected to differentiation. After differentiation, cells were fixed with 4% formalin for 30 min and washed with phosphate-buffered saline. Adipogenic differentiated cells were stained with Oil Red O solution and osteogenic differentiated cells with Alizarin red S solution for 1 h at room temperature. The remaining excessive staining solution was removed, and stained cells were visualized under a microscope.
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