Human GFs were cultured in DMEM (Lonza) supplemented with 10% FBS (Lonza) and 0.1% gentamicin sulfate (10 mg/ml; Euroclone) at 37°C in 5% CO2 atmosphere. Cells between the fourth and tenth passages were used for the following experiments. At Passage 6 cells were seeded overnight with medium containing 1% of FBS (Lonza) and then were treated with or without TGF-β1 for 24, 48, and 72 h. Medium was removed 24 h after seeding and fibroblasts were incubated for 24, 48, and 72 h with DMEM plus with 1% FBS (Lonza) serum alone or supplemented with 10 ng/ml TGF-β1 (S.I.A.L.). Myofibroblasts phenotype was confirmed by flow cytometry, confocal microscopy, and Western blot for α-SMA, Vimentin, E-cadherin, β-catenin, and Smad 2/3. Once the obtained cell differentiation cells were collected to perform all experiments, the untreated cells were used as negative controls (CTRL).
Gentamicin sulfate
Manufactured by Euroclone
Sourced in Italy
Gentamicin sulfate is a broad-spectrum antibiotic commonly used in laboratory settings. It is effective against various types of bacteria, including Gram-positive and Gram-negative strains. Gentamicin sulfate is a critical component in cell culture media and is often used to prevent bacterial contamination in a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Tgf β1, by Lonza (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
Fetal bovine serum (fbs), by Lonza (1 mentions)
Hydrocortisone, by Euroclone (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Euroclone (1 mentions)
Mem neaa, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
F12 medium, by Euroclone (1 mentions)
Sodium pyruvate, by Euroclone (1 mentions)
Fetal bovine serum (fbs), by Euroclone (1 mentions)
L glutamine, by Euroclone (1 mentions)
3 protocols using gentamicin sulfate
1
TGF-β1-induced Myofibroblast Differentiation
2
In Vitro Cytotoxicity Assay for Cancer Cells
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Dulbecco’s phosphate-buffered saline (DPBS) (Euroclone, Milano, Italy); Dulbecco’s Modified Eagle’s Medium (DMEM) (LONZA, BioWhittaker®, Morrisville, NC, USA); Minimum Essential Medium and Non-Essential Amino Acids (MEM, NEAA) (Gibco®, Life Technologies™. Grand Island, NY, USA); endothelial cell basal medium (EBM) (LONZA, Clonetics®, Morrisville, NC, USA); fetal bovine serum (FBS) (Life Technologies. Grand Island, NY, USA); L-glutamine, penicillin, streptomycin. Bovine brain extract (BBE), epidermal growth factor (rhEGF), gentamicin sulfate, ascorbic acid, and hydrocortisone (Euroclone, Milano, Italy); (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction (MTT), cell proliferation assay ATCC® 30-1010K kit (Invitrogen Co). Dimethyl sulfoxide (DMSO) (Sigma Aldrich, St. Louis, MO, USA, ).
The human cancer cells HepG2 (hepatocarcinoma cell line) were obtained from ATCC (American Cell Culture Collection), MCF7 (breast cancer cell line) from the pathological anatomy (Civil hospital of Cagliari) and normal human umbilical vein endothelial cell (HUVEC CC-2519) from LONZA (Clonetics®, Morrisville, NC, USA).
The human cancer cells HepG2 (hepatocarcinoma cell line) were obtained from ATCC (American Cell Culture Collection), MCF7 (breast cancer cell line) from the pathological anatomy (Civil hospital of Cagliari) and normal human umbilical vein endothelial cell (HUVEC CC-2519) from LONZA (Clonetics®, Morrisville, NC, USA).
3
Characterizing BMP6 Peptide Binding in CHO Cells
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Wild-type Chinese Hamster Ovary (CHO-K1 ATCC® CCL-61) and mutant CHO-745 (pgsA-745 ATCC® CRL-2242 [16, 17] ) cells were maintained at 37 • C and 5% CO 2 , in F12 medium (Euroclone) supplemented with 10% Fetal Bovine Serum (FBS, Euroclone), 40 μg/mL gentamicin sulfate (Euroclone), 2 mM L-glutamine (Euroclone) and 1 mM sodium pyruvate (Euroclone). Wild type and mutant CHO cells were seeded in a TC 96-well plate (Sarstedt) with a cellular density of 80.000 cells/well. Once the monolayers formed, the medium was removed, and cells were washed gently with PBS, fixed with 3% glutaraldehyde, incubating for 2 h at 4 • C, while the reaction was stopped adding glycine at a final concentration of 0.1 M. After PBS washing, the non-specific binding sites were saturated with PBS + 3% BSA. Synthetic BMP6 peptides (5, 25, 50, 250 μM in PBS + 3% BSA) were spotted onto cell monolayers and incubated 3 h at 37 • C. Unbound peptides were removed by washing with PBS, while cell-bound peptides were detected as previously described, taking advantage of their biotin tag.
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