MAPCs were isolated from a human donor through bone marrow aspiration. Cell isolation was processed according to previously described methods.25 (link) Briefly, MAPCs were cultured in fibronectin-coated plastic tissue culture flasks. Cell cultures were maintained under low oxygen tension in a humidified atmosphere of 5% CO2. Cells were cultured in a media containing low-glucose (D)MEM (Life Technologies, Grand Island, New York, USA) supplemented with fetal bovine serum (Atlas, Fort Collins, Colorado, USA), ITS liquid media supplement (Sigma), MCDB (Sigma), platelet-derived growth factor (R&D Systems, Minneapolis, Minnesota, USA), epidermal growth factor (R&D Systems), dexamethasone, penicillin/streptomycin (Life Technologies), 2-phospho-L-ascorbic acid and linoleic acid-albumin (Sigma). Cells were passaged every 3–4 days and harvested using trypsin/EDTA (Life Technologies). The cells were positive for CD49c and CD90 and negative for MHC class II and CD45 (all antibodies (Abs) were from BD Biosciences, San Jose, CaliforniaA, USA). Cells were cryopreserved in media and 5% dimethyl sulfoxide. Before administration, the MAPCs were counted with trypan blue exclusion, and the final concentration was adjusted according with the percentage of live cells.
Linoleic acid albumin
Manufactured by Merck Group
Sourced in United States, Germany
Linoleic acid-albumin is a laboratory product manufactured by Merck Group. It is a fatty acid-protein complex commonly used in cell culture and biochemical research applications. The core function of this product is to provide a source of linoleic acid, an essential fatty acid, in a stabilized and soluble form.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Merck Group (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Epidermal growth factor (egf), by R&D Systems (1 mentions)
Dexamethasone (dex), by Thermo Fisher Scientific (1 mentions)
Platelet derived growth factor, by R&D Systems (1 mentions)
2 phospho l ascorbic acid, by Merck Group (1 mentions)
Its liquid media supplement, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
Mem neaa, by Thermo Fisher Scientific (1 mentions)
Advanced dmem, by Thermo Fisher Scientific (1 mentions)
Advanced dmem medium, by Thermo Fisher Scientific (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Antibiotic antimycotic solution, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
6 protocols using linoleic acid albumin
1
Isolation and Characterization of MAPCs
2
Porcine MSCs Adipogenic Differentiation
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Porcine mesenchymal stem cells were isolated from adipose tissue and bone marrow from a 3-month-old Polish Large White pig, as described by Kociucka et al. (2016 (link)). The cells were cultured in advanced DMEM medium (Gibco) supplemented with 10% FBS (Sigma-Aldrich), 5 ng/ml FGF-2 (PromoKine), 2 mM L-Glutamine (PAA), 1 mM 2-mercaptoethanol (Sigma-Aldrich), 1 × antibiotic antimycotic solution (Sigma Aldrich), and MEM NEAA (Thermo Fisher Scientific) at 37 °C with 5% CO2 supplementation. To induce adipogenic differentiation, the cells were grown to confluency and were cultured with adipogenic differentiation medium composed of advanced DMEM (Gibco) with 10% (v/v) FBS (Sigma), 1 × antibiotic antimycotic solution (Sigma Aldrich), MEM NEAA (Thermo Fisher), 5 ng FGF-2 (PromoKine), 1 × linoleic acid albumin (Sigma-Aldrich), 1 × ITS (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), 100 μM indomethacin (Sigma-Aldrich), and 50 mM IBMX (Sigma-Aldrich). The differentiation process was allowed to proceed for 7 days and lipid droplet formation was examined using a phase-contrast microscope (TS100 Eclipse, Nikon).
3
Culturing Medulloblastoma and Granule Cell Progenitor Cells
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Medulloblastoma cell lines DAOY and ONS-76 were cultured in MEM and RPMI 1640 medium (GIBCO), respectively, supplemented with 10% fetal bovine serum (GIBCO), 100 units/mL penicillin, and 100 μg/mL streptomycin (Invitrogen), 1.25 µg/ml fungizone (Invitrogen). DAOY Medium was supplemented with 0.1 mM non-essential amino acids and 1 mM sodium pyruvate. Granule cell progenitors (GCP) were purified from mouse cerebella dissected at P7. Following dissociation (30 min, 37 °C) in trypsin/DNAse solution at 37 °C and trituration in DNAse, cells were separated on a density step gradient of 35% and 60% Percoll solution (Sigma). Purified GCPs were further enriched by panning on tissue culture dishes to remove adherent fibroblasts. Non adherent cells were plated at a density of 4 × 106 cells/mL in 12 wells plate coated with a poly D-lysine solution (Sigma) and Matrigel (BD Biosciences), with 12.5 μg/mL SHH (R&D). Cells were grown in Neurobasal medium with B27 supplement, 2 mM glutamine, and 100 U/mL penicillin/streptomycin (all from Invitrogen), linoleic acid-albumin, 0.45% D-glucose, and 16 μg/mL N-acetyl-cysteine, 1% SPITE (all from Sigma-Aldrich). After 1 h plating, GCPs were infected, washed and cultured (37 °C, 5% CO2) in their medium for 48 h when their RNA was extracted.
4
Cholangiocyte Differentiation from hESCs
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hESC were differentiated into cholangiocytes as described by Dianat et al. [34 (link)].
The biliary cysts were generated as follows: day 18 cells were detached with trypsin, centrifuged and resuspended in biliary differentiation media (BDM; William’s E/Ham F12 1:1, 10−5 M linoleic acid-albumin (Sigma L9530), 5 × 10−8 M 3,3′,5-triiodo-L-thyronine (Sigma T2752), 0.2 IU insulin, 6.10−4 M vitamin C (Boyer), 6 × 10−4 M human apo-transferrin (Sigma T5391), 1 mM sodium pyruvate (Gibco)). The cells were then added to a mixture of rat-tail type I collagen (BD Biosciences), Matrigel 40 % (BD Biosciences), HEPES (0.02 M) and NaHCO3 (2.35 mg/ml). A cell suspension containing 5000 cells was added to 24-well inserts (BD biosciences 353104) and incubated at 37 °C for 3 hours. BDM medium (1 ml) supplemented with 20 ng/ml HGF and 10 ng/ml epidermal growth factor (EGF) was added on top of the insert as well as in the well, and incubated at 37 °C/5 % CO2 up to 2 weeks. The cells were fed every other day and, after 2 weeks, the cells were fixed in 3 % paraformaldehyde and stained.
The biliary cysts were generated as follows: day 18 cells were detached with trypsin, centrifuged and resuspended in biliary differentiation media (BDM; William’s E/Ham F12 1:1, 10−5 M linoleic acid-albumin (Sigma L9530), 5 × 10−8 M 3,3′,5-triiodo-L-thyronine (Sigma T2752), 0.2 IU insulin, 6.10−4 M vitamin C (Boyer), 6 × 10−4 M human apo-transferrin (Sigma T5391), 1 mM sodium pyruvate (Gibco)). The cells were then added to a mixture of rat-tail type I collagen (BD Biosciences), Matrigel 40 % (BD Biosciences), HEPES (0.02 M) and NaHCO3 (2.35 mg/ml). A cell suspension containing 5000 cells was added to 24-well inserts (BD biosciences 353104) and incubated at 37 °C for 3 hours. BDM medium (1 ml) supplemented with 20 ng/ml HGF and 10 ng/ml epidermal growth factor (EGF) was added on top of the insert as well as in the well, and incubated at 37 °C/5 % CO2 up to 2 weeks. The cells were fed every other day and, after 2 weeks, the cells were fixed in 3 % paraformaldehyde and stained.
5
Preparation of Palmitate-BSA Complex
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palmitate-BSA stock was prepared at a final concentration of 4 mM palmitate with 12% fatty acid-free BSA (molecular ratio 2.2:1) (both purchased from Sigma-Aldrich) in glucose/pyruvate-free Krebs-Henseleit buffer (KHB) with modifications as described previously (Bakrania et al., 2016 ). In brief, palmitate was dissolved in 38% ethanol in 0.5 mM Na2CO3 at 60°C under constant nitrogen gas. Upon dissolving, ethanol was boiled off by increasing the temperature to 80°C–90°C. BSA was dissolved in KHB at 37°C. BSA solution and palmitate solution were mixed together under constant stirring at 37°C. Successful complexing of palmitate to BSA was monitored when no clouding of the solution occurred. The BSA-palmitate solution was stirred for an additional 5 min before being dialyzed overnight and sterile filtered using 0.45-μm pore size membrane filter. Aliquots were stored at −20°C. Oleic acid-albumin and linOleic acid-albumin complexes were purchased from Sigma.
6
Serum-free cell culture media protocols
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All media including commercial serum-free medium for static culture (SFS), commercial serum-free medium for dynamic culture 1 (SFD1) and commercial serum-free medium for dynamic culture 2 (SFD2) were purchased from Gibco, Gaithersburg, MD, USA and DMEM-Ham's/F12 was from Sigma-Aldrich, St Louis, MO, USA. All chemical supplements including 100X concentrated hypoxanthinethymidine (HT) supplement was obtained from Gibco, USA, 100 mM L-methionine sulfoximine (MSX) was from Millipore, Burlington, MA, USA recombinant human insulin and NEAA mixture were from Biochrome, Berlin, Germany plant peptone, linoleic acid-albumin, yeast extract, L-glutamine, recombinant human transferrin and BSA were from Sigma, USA and dextran sulphate and Pluronic F68 were from Applichem, Darmstadt, Germany. Bambanker™ serum-free type cell freezing medium was obtained from Lymphotec Inc., Tokyo, Japan. After this section, medium with 1% (v/v) HT supplement and 0.05% (v/v) MSX is called as ready-to-use medium for this study.
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