8 × 2 mm acellular hydrogels (n = 4 per group) were fabricated as described above. The hydrogels were incubated in complete osteogenic medium without fetal bovine serum (FBS), complete osteogenic medium with 10% FBS (Cambrex Bioscience), PBS pH 7.4, and 1× simulated body fluid for 0, 1, 7, 14, and 28 days at 37°C in 12-well plates. The simulated body fluid was prepared according to published protocols [30 (link)]. Each solution was changed every 2-3 days. At timepoints, the hydrogels were soaked in ultrapure water for 30 min, cut in half, blotted and weighed. Sample halves (n = 4 halves) for the calcium assay were placed in 500 μL of ddH2O, homogenized through three freeze/thaw/sonication cycles, and digested overnight in equal parts of 1N acetic acid for a final concentration of 0.5 N acetic acid. The assay was performed with a commercially available kit (Sekisui Diagnostics, Tokyo, Japan) according to the manufacturer’s instructions. The remaining sample halves were fixed in 10% formalin, dehydrated in 70% ethanol, placed in Histoprep frozen tissue embedding media (Fischer Scientific, Waltham, MA), and frozen at −20°C.
Fetal bovine serum (fbs)
Manufactured by Cambrex
Sourced in United States, Switzerland, Belgium, Italy, United Kingdom
Fetal bovine serum is a cell culture media supplement derived from the blood of bovine fetuses. It provides a rich source of proteins, growth factors, and other nutrients essential for the growth and proliferation of cells in in vitro cell culture systems.
Automatically generated - may contain errors
Lab products found in correlation
Streptomycin, by Merck Group (8 mentions)
Penicillin, by Merck Group (8 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Penicillin, by Cambrex (6 mentions)
Streptomycin, by Cambrex (6 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Glutamax, by Thermo Fisher Scientific (5 mentions)
L glutamine, by Merck Group (5 mentions)
Trypsin edta, by Merck Group (4 mentions)
Streptomycin, by Thermo Fisher Scientific (4 mentions)
Penicillin, by Thermo Fisher Scientific (4 mentions)
Glutamine, by Cambrex (4 mentions)
Rpmi 1640, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Glutaraldehyde, by Merck Group (2 mentions)
Methanol, by Avantor (2 mentions)
Chloroform, by Avantor (2 mentions)
Diethyl ether, by Avantor (2 mentions)
Acetonitrile, by Avantor (2 mentions)
Super q water system, by Merck Group (2 mentions)
61 protocols using fetal bovine serum (fbs)
1
Acellular Hydrogel Mineralization Kinetics
2
Culturing HDF Cells for Experiments
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HDF cells were purchased from American Type Culture Collection (catalog no. PCS-201-010) and maintained at 37°C with 5% CO2 and >90% humidity inside the incubator. Passages 1 to 6 are used in the experiment, and no significant difference in the behavior of different passages is recorded. Cell culture medium is composed of Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 10% fetal bovine serum (FBS) (Clonetics), GlutaMAX, and penicillin/streptomycin (both from Thermo Fisher Scientific). The cells were plated at the surface density 3.3 × 107 m−2 (fixed for all experiments) onto the substrates placed in a Petri dish made of a sterilized glass [to allow for the polarized optical microscopy (POM) imaging].
3
Culturing Human Microvascular Endothelial Cells
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Human microvascular endothelial cells were purchased from Lonza (catalog #: CC-2543; Allendale, NJ, USA). Cells were cultured in endothelial growth medium (EGM™-2MV BulletKit™; Clonetics) supplemented with 5% fetal bovine serum (FBS; Clonetics) and incubated at 37°C and 5% CO2 in cell culture incubator. For continuous passaging, the cells were trypsinized and dissociated, then were centrifuged at 220 × g for 7 min, diluted, and re-plated at the appropriate densities. All cells were used before passage 4.
4
Bovine Angiogenin Induces Vascular Development
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Example 2
Angiogenin was provided in an enriched extract prepared from bovine skim-milk according to the method described above.
An angiogenesis assay employing human umbilical vein endothelial cells (HUVECS) was used to determine if bovine angiogenin is active on human cells. HUVEC cells were routinely maintained in Endothelial cell basal (ECB) medium, supplemented with bovine brain extract, EGF, hydrocortisone and 10% FBS (Clonetics). Assays were performed in triplicate in 48 well tissue culture plates. 150 μl of Matrigel (BD biosciences) was first allowed to polymerise on the bottom of each well. HUVEC cells were resuspended in ECB with now 1% FBS and bovine angiogenin, at 0.5×106 cells/ml. The cells (2.5×104 cells/well) were then plated on to the Matrigel matrix and incubated at 37° C. for 24 hours. Human vascular endothelia growth factor (VEGF) 10 ng/ml replaced angiogenin as a positive control and ECB media 1% FBS alone was used a negative control. Vascular development was observed and photographed at 10× magnification and the results shown in
The results show that bovine angiogenin induces vascular development of HUVEC on Matrigel in the same manor as human VEGF and therefore bovine angiogenin is shown to be active on human cells.
5
Culturing Human Pulmonary Artery Endothelial Cells
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Human pulmonary artery endothelial cells (ECs) were cultured in essential growth medium (EGM-2) containing 10% fetal bovine serum (Clonetics, Walkersville, MD, USA). Cells were placed in an incubator at 37 °C, 5% CO2, and 95% humidity to achieve contact-inhibited monolayers.
6
Hypoxic Cultivation of Human Hepatocellular Carcinoma Cells
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The human HCC cells HAK1-B and KYN-2 were provided by the Department of Pathology, Kurume University School of Medicine, while Huh-7 cells were obtained from Cambrex (Walkersville, MD). Cells were maintained in Dulbecco modified Eagle medium (Gibco Invitrogen Cell Culture, Auckland, New Zealand) supplemented with 10% fetal bovine serum (Biowest, Nuaille, France). Human umbilical vascular endothelial cells were purchased from Cambrex and maintained in endothelial cell growth medium-2 (Clonetics, San Diego, CA) containing 10% fetal bovine serum. All cell lines were maintained at 37 °C in a humidified atmosphere with 5% CO2. For in vitro hypoxic assays, cells were cultivated in hypoxic conditions that were maintained by a hypoxia chamber (APM-30D, ASTEC, Fukuoka, Japan) flushed with a gas mixture of 3% O2, 5% CO2, and 92% N2.
7
Culturing Human Pulmonary Artery Endothelial Cells
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Human pulmonary artery endothelial cells (EC) were cultured in an essential growth medium (EGM-2) containing 10% fetal bovine serum (Clonetics, Walkersville, MD, USA). EC was then allowed to grow to achieve confluent monolayers in an incubator at 37 °C, 5% CO2, and 95% humidity.
8
Osteogenic Hydrogel Scaffold Synthesis
9
Evaluation of DNA Damage and Apoptosis
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The deoxycytidine kinase kit was obtained from EIAab (Wuhan, China, Cat no: E8892h). Human H2A.X (phospho S139) In-Cell ELISA Kit was purchase in abcam (Cat no: ab131382). CellEvent™ Caspase-3/7 Green Detection Reagent was from Thermo Fisher Scientifc (Cat no: C10423). The Fluo-4 NW Calcium Assay Kit was obtained from Thermo Fischer Scientific (Waltham, MA, USA, Cat no: F36206). Culture medium (RPMI 1640) and fetal bovine serum (FBS) were obtained from Cambrex (Basel, Switzerland); trypsin-EDTA, penicillin, streptomycin, and VE-821 (Cat no: SML1415) were acquired from Sigma-Aldrich (St. Louis, MO, USA). Other chemicals and solvents were of high analytical grade and were obtained from Sigma-Aldrich or Avantor Performance Materials Poland S.A. (Gliwice, Poland).
10
Multiparametric Cellular Assay Protocol
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Acridine orange, 2,3-diaminonaphthalene (DAN), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazol-carbocyanine iodide (JC-1), 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA), and Autophagy Assay kit were obtained from Sigma Aldrich cat. no.:MAK138 (St. Louis, Missouri, USA). Human MAP1LC3A (Microtubule-associated proteins1A/1B light chain 3A) ELISA Kit was obtained from Fine-test (Wuhan, Hubei, China). The Fluo-4 NW Calcium Assay Kit was obtained from Molecular Probes (Eugene, USA). The Sphingomyelinase Assay kit was purchased from Abcam (Cambridge, UK). Culture media (DMEM, RPMI 1640) and fetal bovine serum (FBS) were obtained from Cambrex (Basel, Switzerland); trypsin-EDTA, penicillin and streptomycin were acquired from Sigma Aldrich (St. Louis, Missouri, USA).
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