ASC differentiation was performed as reported [19 (link)]. To induce adipogenic differentiation, ASCs were cultured with StemMACS AdipoDiff media (Miltenyi Biotec, Gladbach) up to 14 days. Cells were then fixed and stained for oil red O and adiponectin (Abcam, Cambridge, #AB22554) characteristic of adipocytes. For osteogenic differentiation, ASCs were incubated in StemMACS OsteoDiff media (Miltenyi Biotec) up to 14 days, fixed, and stained with 2% alizarin red S (pH 4.2) to visualize calcific deposition in cells of an osteogenic lineage. Western blot analysis was performed as reported [21 (link), 24 (link)], using rabbit monoclonal antibodies against p44/42 Erk1/2 (#9102), rabbit polyclonal phospho-p44/42 Erk1/2 (Thr202/Tyr204) (#9101), rabbit monoclonal GSK3β (27C10) (#9315), rabbit polyclonal phospho-GSK3β (Ser 9) (#9331), mouse monoclonal STAT3 (124H6) (#9139), rabbit monoclonal pSTAT3 (Tyr705) (#9313), mouse monoclonal β-actin (A2228) (Sigma-Aldrich), and GAPDH (#MA5-15738) from ThermoFisher Scientific (Frankfurt).
Stemmacs adipodiff media
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
StemMACS AdipoDiff Media is a cell culture medium specifically designed for the differentiation of human mesenchymal stem cells (hMSCs) into adipocytes. The medium provides the necessary components to support the in vitro differentiation of hMSCs into mature adipocytes.
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Lab products found in correlation
Stemmacs osteodiff media, by Miltenyi Biotec (6 mentions)
Oil red o solution, by Merck Group (2 mentions)
Stemmacs chondrodiff media, by Miltenyi Biotec (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Oil red o, by Merck Group (2 mentions)
Alcian blue, by Merck Group (2 mentions)
Mouse monoclonal β actin a2228, by Merck Group (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Dmem f12 media, by Merck Group (1 mentions)
Bcip nbt, by Merck Group (1 mentions)
Stemxvivo chondrogenic supplement, by R&D Systems (1 mentions)
Anti aggrecan, by R&D Systems (1 mentions)
Oil red o, by Abcam (1 mentions)
Osteodiff media, by Miltenyi Biotec (1 mentions)
Alizarin red s, by Merck Group (1 mentions)
Sb431542, by Miltenyi Biotec (1 mentions)
Stemmacs ips brew xf, by Miltenyi Biotec (1 mentions)
Msc expansion medium, by Miltenyi Biotec (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
α mem, by Thermo Fisher Scientific (1 mentions)
12 protocols using stemmacs adipodiff media
1
Adipogenic and Osteogenic Differentiation of ASCs
2
Adipocyte Differentiation Assay
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Cells were plated at 7500 cells/cm2 in 6-well plates and induced to differentiate in the StemMACS™AdipoDiff Media (Miltenyi Biotec, Bergisch Gladbach, Germany) differentiation medium for 21 days. Cells cultivated in basal medium (D-MEM, low glucose with 10% FBS) were used as a control. Differentiation and basal medium were changed every 48–72 h and cells were examined by inverted microscopy. The formation of lipid droplets was verified with Oil Red O staining.
3
Multilineage Differentiation Potential of Adipose-Derived Stem Cells
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The differentiation capacity was assessed following the manufacturer's instructions for commercial differentiation kits. For rADSCs, cells were cultured in a differentiation medium (StemXVivo Osteogenic/Adipogenic Media-R&D Systems, Minneapolis, MI, USA) with adipogenic or osteogenic supplements contained in the kit. Adipocytes were detected by Oil Red O (Sigma-Aldrich, Saint Louis, MO, USA) staining and osteoblasts by NBT/BCIP (Sigma-Aldrich, Saint Louis, MO, USA) staining. To differentiate into chondrocytes, 2.5 × 105 cells were cultured in DMEM/F-12 media (Sigma-Aldrich, Saint Louis, MO, USA) plus chondrogenic supplements (StemXVivo Chondrogenic Supplement-R&D Systems) in the bottom of a 15 ml conical tube. The cell pellet formed a rounded ball approximately 1-2 mm in diameter on the 3rd day. On the 14-28th days, the pellet was fixed in 4% paraformaldehyde and cut into 10 μm sections. They were treated with chondrocyte-specific primary antibody anti-aggrecan (R&D Systems, Minneapolis, MI, USA) and anti-rabbit Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MS, USA) as a secondary antibody. For hADSCs, cells were cultured in StemMACS AdipoDiff media and StemMACS OsteoDiff media (Miltenyi Biotec, Bergisch Gladbach, Germany). The procedure of detection was similar to that of rADSCs.
4
Adipogenic Differentiation of Mesenchymatic Stromal Cells
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Mesenchymal stromal cells were cultured with DMEM and passaged twice/three times. Then, cells were seeded into 12-well plates, and adipogenic induction was performed using StemMACS™ AdipoDiff Media (Miltenyi Biotec). Cells were cultured in presence of complete adipogenic medium or with 70% AdipoDiff Media plus 30% DC-CM or DC/MSC-CM or 30% basal medium, or recombinant human OPN (1 µg/ml) (Peprotech). Medium was changed every 4/5 days and mRNA extraction was performed at 5 and 12 days while lipid droplet staining was evaluated at 15 days of culture. In some experiments, cells cultured in presence of DC-CM were treated with neutralizing monoclonal antibodies against CD44 (clone 5F12; Lifespan Biosciences, Inc.) and CD29 (clone P5D2; R&D Systems) or with the corresponding isotype control antibody at 10 µg/ml (R&D Systems).
5
Adipogenic Differentiation of Cells
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A total of 5000 cells/well were plated in a 24-well plate with culture medium. After 5 days, the medium was removed and replaced with adipogenic medium (StemMACS AdipoDiff Media, Miltenyi Biotec). Cells were fed weekly with complete replacement of adipogenic medium. On day 7, cells were stained with Oil Red O solution (Sigma) after fixation (8% formaldehyde). Lipid vacuoles were then observed by light microscopy.
6
Differentiation of WJ-MSCs into Adipocytes, Osteoblasts, and Chondrocytes
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For differentiation into adipocytes and osteoblasts, WJ-MSCs cultured in FBS and XF conditions were plated in 6-well plates at a concentration of 1 × 105 cells. After the cells reached 70–80% confluency, they were treated with StemMACS™ Adipodiff Media and OsteoDiff Media (Miltenyi Biotec, Bergisch Gladbach, Germany), following the manufacturer’s instructions. The medium was changed every 3 days. After 3 weeks of induction, Oil Red O (Abcam, Cambridge, UK) staining or Alizarin Red S (Sigma-Aldrich, St. Louis, MO) staining was performed to evaluate lipid-laden fat cells or calcium deposition, respectively. For chondrogenic differentiation, 2.5 × 105 cells were placed in a 15-mL polypropylene tube and maintained with 1 mL of StemMACS™ Chondrodiff Media (Miltenyi Biotec), following the manufacturer’s instructions. The cells were allowed to undergo differentiation for 3 weeks, with media change every 3 days. After differentiation, the round pellets were embedded in paraffin and cut into 3-µm sections that were stained by Alcian Blue (Merk Millipore, Darmstadt, Germany) to detect glycosaminoglycans.
7
Adipogenic and Osteogenic Differentiation of ASCs
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ASC differentiation was performed as reported (Ritter et al., 2015 (link)). To induce adipogenic differentiation, ASCs were cultured with StemMACS AdipoDiff media (Miltenyi Biotec, Gladbach) up to 14 days. Cells were then fixed and stained for oil red O and adiponectin (Abcam, Cambridge, #AB22554) characteristic of adipocytes. For osteogenic differentiation, ASCs were incubated in StemMACS OsteoDiff media (Miltenyi Biotec) up to 21 days, fixed, and stained with 2% alizarin red S (pH 4.2) to visualize calcific deposition by cells of an osteogenic lineage.
Western blot analysis was performed as reported (Muschol-Steinmetz et al., 2016 (link), Steinhauser et al., 2017 (link)), using mouse monoclonal antibodies against cyclin B1 (#sc-752) and β-actin (#sc-47778) from Santa Cruz Biotechnology (Heidelberg).
Western blot analysis was performed as reported (Muschol-Steinmetz et al., 2016 (link), Steinhauser et al., 2017 (link)), using mouse monoclonal antibodies against cyclin B1 (#sc-752) and β-actin (#sc-47778) from Santa Cruz Biotechnology (Heidelberg).
8
Mesenchymal Stem Cell Differentiation
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MSC differentiation was induced by exchanging StemMACS iPS-Brew XF medium with differentiation medium containing 20% KSR, 1% GlutaMAX, 1% NEAA, and 0.1 mM beta-mercaptoethanol supplemented with 10 μM SB431542 (Miltenyi Biotec). Fresh medium was applied every other day and after 7 days, cells were transferred in a 1:3 ratio to a non-coated tissue culture-treated plate with MSC expansion medium (Miltenyi Biotec). Fresh medium was applied daily before splitting the cells at differentiation day 14. Process control of MSC differentiation was performed by flow cytometry and RT-qPCR at day 21. At day 21, MSCs were differentiated to adipocytes or osteocytes using StemMACS AdipoDiff Media or StemMACS OsteoDiff Media (both Miltenyi Biotec), respectively. Fresh medium was applied every 3 days for 20 days before process control by OilRed O or Alizarin Red staining, respectively.
9
Adipocyte and Osteoblast Differentiation in Vitro
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2.104 cells/well were cultured in 24- or 6-well culture plates, with complete Minimum Essential Medium α (MEMα) (Thermofisher, Waltham, MA, USA) or with StemMACS AdipoDiff Media or StemMACS OsteoDiff Media (Miltenyi Biotec, Bergisch Gladbach, Germany), for adipocyte and osteoblast differentiation, respectively. The medium was changed twice a week. All 24-well cultures were stopped after 21 days, for colorimetric testing. Alizarin red staining—the cells were fixed with 70% ethanol, stained with 2% Alizarin Red for 10 min, washed with H2O, and analyzed. For quantification, the plates were thawed, distained by the addition of 800 μL of 10% acetic acid chloride monohydrate. The optical density was then measured at OD405 and the relative ratio of the cells cultured in the osteogenic conditions were determined, relative to the cells cultured in the stromal medium. [21 (link)]. For adipocyte differentiation, the Nile red (Sigma-aldrich, Missouri, USA) stain was assessed according to the manufacturer’s guidelines. Cultures in the 6-well plates were stopped after 14 days for the RT-PCR tests and the cells were harvested using trypsin with EDTA (Invitrogen, Carlsbad, CA, USA).
10
Adipogenic Differentiation Assay
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A total of 5000 cells/well were plated in a 24-well plate with classical culture medium. After 5 days, the medium was replaced with adipogenic medium (StemMACS AdipoDiff Media, Miltenyi Biotec). Cells were fed weekly with complete replacement of adipogenic medium. On Day 7, the cells were stained with Oil Red O solution (Sigma) after fixation (8% formaldehyde). Lipid vacuoles were then observed by light microscopy.
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