MSCs were obtained from young (n: 11) and older (n: 6) bone-marrow donors. After acquiring informed consent, bone-marrow aspirates were obtained from the young patients and, from a second group, bone scrapings were obtained following hip replacement surgery. Mononuclear cells were isolated by Ficoll density centrifugation (400g, 25 min, 20 °C), washed twice by sedimentation with phosphate buffer (300g, 5 min) and the cells re-suspended in MSC medium (DMEM plus 10 % FBS) and seeded into culture flasks (Nunc, Roskilde, Denmark) at 1.5 × 105 cells/cm2 and allowed to adhere for 24 h. MSCs were then cultured (37 °C, 5 % CO2) in MSC medium. DNA methylation and hydroxymethylation analyses were carried out at cell passages 4–7 (Additional file 1 : Table S1). The study was approved by the Ethics Committee of Clinical Research at Hospital Universitario Niño Jesús and Hospital U.M. Valdecilla, and written informed consent was obtained from patients and parents/tutors.
Culture flasks
Manufactured by Thermo Fisher Scientific
Sourced in Denmark
Culture flasks are laboratory equipment used to cultivate cells or microorganisms in a controlled environment. They provide a sterile, contained space for growing and maintaining cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Hep3b, by National Centre for Cell Science (1 mentions)
Hepg2, by Thermo Fisher Scientific (1 mentions)
Hepg2, by National Centre for Cell Science (1 mentions)
Mcf 7, by RIKEN BioResource Center (1 mentions)
Mcf 7, by Olympus (1 mentions)
Zr 75 1, by RIKEN BioResource Center (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Trypsin, by Merck Group (1 mentions)
Collagenase type 2, by Worthington (1 mentions)
Mem non essential amino acid solution, by Merck Group (1 mentions)
Trypsin edta, by Thermo Fisher Scientific (1 mentions)
L proline, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Merck Group (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
12 protocols using culture flasks
1
Epigenetic Profiling of Mesenchymal Stem Cells
2
Human Dermal Fibroblast Extraction and Culture
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The lines of human dermal fibroblasts were obtained from donor skin, as described previously [10 (link),13 (link),14 (link)]. Briefly, tissue biopsies were collected from patients (who have given the informed contest) during surgery. The study was approved by the Ethics Committee of the Institute of Medical Cell Technologies, Ekaterinburg. Skin biopsies were cut into small pieces, and cells were extracted by tissue dissociation method. Extracted fibroblasts were grown in culture flasks (Nunc, Roskilde, Denmark) at 37 °C and 5% CO2. Cells were passaged when they covered 80% of the flask surface area. Then sub-culturing cells were treated with 0.25% trypsin-EDTA solution (Gibco, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Cell number was estimated by cell counter TC-20 device (Bio-rad, Hercules, CA, USA). The cell viability was measured by staining with trypan blue. Fibroblasts were stored in liquid nitrogen.
3
Isolation and Expansion of Mesenchymal Stem Cells
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MSCs were purchased from Lonza (Verviers), Millipore (Billerica), and Inbiobank or directly obtained from young and elderly donors. After informed consent, bone marrow aspirates were obtained from one group of patients, and from a second group, bone scrapings were obtained following hip replacement surgery. Mononuclear cells were isolated by Ficoll density centrifugation (400 g, 25 min, 20°C) and washed twice by sedimentation with phosphate buffer (300 g, 5 min), and the cells were resuspended in MSC medium (DMEM plus 10% FBS) and seeded into culture flasks (Nunc) at 1.5 × 10−5 cells/cm2 and allowed to adhere for 24 h. MSCs were then cultured (37C, 5% CO2) in MSC medium. DNA methylation analyses were carried out at cell passages 4–6 (Supplemental Table 1).
4
Cell Line Cultivation and Characterization
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The human hepatoma (HepG2, Hep3B, and Huh7) cell lines were purchased from the National Centre for Cell Science (NCCS), Pune, India. Primary hepatocytes were procured from American Type Culture Collection (ATCC), USA. HepG2, Hep3B and Huh7 cells were grown in culture flasks (Nunclon, Denmark) and maintained in DMEM supplemented with 10% FBS and 1% PenStrep solution in a humidified 5% CO2 incubator at 37 °C. For the cultivation of primary hepatocyte (THLE-2) cells, DMEM/F-12 media were used. The final growth medium consisted of the following: DMEM/F-12 with 10% FBS, 5 ng/mL EGF and 70 ng/mL phosphoethanolamine. THLE-2 cells require a special coating medium, which consists of the following: DMEM/F-12 without glutamine supplemented with BSA (heat shock fraction) and type I collagen peptide. Before seeding the coating, the medium was aspirated. Sub-confluent cells were harvested using 0.25% trypsin–EDTA and cells were re-suspended in complete media and counted using a haemocytometer. Each experimental data point represents the mean of triplicate wells from three independent experiments.
5
Generation of DCs from Blood Monocytes
6
Comparative Morphology of Breast Cancer Cell Lines
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MCF-7 and ZR-75-1 cells were purchased from Bioresource Collection and Research Center (Food Industry Research and Development Institute, Taiwan) and grown in culture flasks (Nunc, Roskilde, Denmark) containing RPMI1640, with 10% heat-inactivated fetal bovine serum (FBS) and streptomycin sulfate, in a 5% CO2 atmosphere at 37 °C until cells were sub-confluent. After incubation, morphological comparison between MCF-7 and ZR-75-1 cells was observed by using the Olympus microscope (IX71) at 20× fitted with an ocular grid.
7
Huh7.5 Cell Culture and Maintenance
8
Bovine Chondrocyte Isolation and Culture
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Bovine chondrocytes
were isolated from the knee of 10- to 12-month-old calves obtained
from the local abattoir. Cartilage pieces were collected from the
condyles, minced, and kept overnight in digestion medium containing
150 U/mL type 2 collagenase (Worthington) in DMEM with 10% heat inactivated
foetal bovine serum (FBS, Sigma-Aldrich), 100 U/mL penicillin, and
100 μg/mL streptomycin (Gibco). The cells were filtered through
a 200 μm cell strainer, washed 3 times with PBS, and stored
at −80 °C in proliferation medium with 20% FBS and 10%
dimethyl sulfoxide (Sigma-Aldrich). Chondrocyte proliferation medium
consisted of DMEM with 10% FBS, 100 U/mL penicillin and 100 μg/mL
streptomycin, 20 μM ascorbic acid (Sigma-Aldrich), 40 ug/mLl -proline (Sigma-Aldrich), and 1% MEM nonessential amino acid
solution (Sigma-Aldrich).
Cells were seeded at 2500 cells/cm2 in culture flasks (Nunc) with chondrocyte proliferation medium
and cultured in hypoxic conditions: 37 °C, 95% humidity, 5% CO2, and 2.5% O2. At 80% confluency, cells were trypsinized
with 0.25% trypsin–EDTA (Gibco). Cells were used at passage
1, and a new donor was used for each experiment.
were isolated from the knee of 10- to 12-month-old calves obtained
from the local abattoir. Cartilage pieces were collected from the
condyles, minced, and kept overnight in digestion medium containing
150 U/mL type 2 collagenase (Worthington) in DMEM with 10% heat inactivated
foetal bovine serum (FBS, Sigma-Aldrich), 100 U/mL penicillin, and
100 μg/mL streptomycin (Gibco). The cells were filtered through
a 200 μm cell strainer, washed 3 times with PBS, and stored
at −80 °C in proliferation medium with 20% FBS and 10%
dimethyl sulfoxide (Sigma-Aldrich). Chondrocyte proliferation medium
consisted of DMEM with 10% FBS, 100 U/mL penicillin and 100 μg/mL
streptomycin, 20 μM ascorbic acid (Sigma-Aldrich), 40 ug/mL
solution (Sigma-Aldrich).
Cells were seeded at 2500 cells/cm2 in culture flasks (Nunc) with chondrocyte proliferation medium
and cultured in hypoxic conditions: 37 °C, 95% humidity, 5% CO2, and 2.5% O2. At 80% confluency, cells were trypsinized
with 0.25% trypsin–EDTA (Gibco). Cells were used at passage
1, and a new donor was used for each experiment.
9
Human Fetal Hepatocyte 3D Culture
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WRL 68 cell line (ATCC, USA) of human fetal hepatocytes was cultured in EMEM medium (BioWhittaker, Cambrex, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Invitrogen USA), glutamine, penicillin 100 U/ml and streptomycin 100 µg/ml (Sigma-Aldrich, Czech Republic) at 37 °C in a humid atmosphere of 95% air and 5% carbon dioxide. WRL 68 cells were grown in a monolayer or 3D cultures. A monolayer was obtained by growing the cells in culture flasks (Nunc, Denmark) and passaging them twice per week (split ratio 1 : 15). Cells in a monolayer were grown for 9 days only. For a long-term (12 weeks) and 3D growth in vitro, 250,000 cells were cultured on a special 25-µm thick plastic bottom membrane in PetriPerm dishes (Vivascience, Germany) that is well permeable to oxygen and carbon dioxide. The culture medium was changed three times a week.
The viability test was performed with propidium iodide 50 µg/ml added to the culture for 10 min after a thorough washing of cells with PBS under TE 300 Eclipse (Nikon, Japan) equipped with epifluorescence.
The viability test was performed with propidium iodide 50 µg/ml added to the culture for 10 min after a thorough washing of cells with PBS under TE 300 Eclipse (Nikon, Japan) equipped with epifluorescence.
10
Bovine Satellite Cell Culture Protocol
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96-well plates (Greiner Bio-one, Netherlands) and culture flasks (Thermo Fisher Scientific, Netherlands) were coated with 0.25 μg/cm2 bovine collagen type I (Sigma-Aldrich, Netherlands) and incubated for at least 1 h in a humidified incubator (37°C, 5% CO2). Prior to use, the plates and culture flasks were washed twice with PBS. After thawing, FACS sorted satellite cells were seeded at a minimum density of 1,800 cells/cm2 or higher densities as indicated. For serial passaging, cells were passaged to maintain a density of <80% confluence and counted at each passage. Where indicated, bovine satellite cell differentiation was induced when at 90%–95% confluence with DMEM (1 g/L glucose) + 2% FBS.
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