For co-culture experiments, pericytes were initially seeded on 60-mm gelatin-coated petri dishes and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies) supplemented with 20% (v/v) FBS (Life Technologies), 2 mM L-glutamine, 50 µg/mL gentamycin and 1 ng/mL bFGF. The cells reached confluency after 2 days. 45×103 cells were seeded into each well of 12-well plates (Costar). CD34+-ECs growing on gelatin-coated 100 mm petri dishes in EGM-2 (with all the supplements except FBS and gentamycin/amphotericin) supplemented with 2% (v/v) FBS, 50 µg/mL gentamycin (Biochrom AG) and 1 ng/mL bFGF were trypsinized and cells were seeded at a density of 8×104/insert onto the Matrigel-coated (BD Biosciences) Transwell inserts (Costar).
Gentamycin
Manufactured by Harvard Bioscience
Sourced in Germany, United States
Gentamycin is a type of antibiotic used in laboratory settings. It is a protein synthesis inhibitor that targets the 30S ribosomal subunit of bacterial cells.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
L glutamine, by Harvard Bioscience (4 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Vegf165, by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (4 mentions)
Rpmi 1640, by Harvard Bioscience (3 mentions)
Penicillin streptomycin, by Harvard Bioscience (3 mentions)
Ecm basal medium, by ScienCell (3 mentions)
Matrigel coated, by BD (3 mentions)
Transwell insert, by Corning (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Fbs superior, by Harvard Bioscience (2 mentions)
Amphotericin, by Harvard Bioscience (2 mentions)
Penicillin, by Harvard Bioscience (2 mentions)
Streptomycin, by Harvard Bioscience (2 mentions)
Endothelial cell growth supplement (ecgs), by ScienCell (2 mentions)
Penicillin streptomycin solution, by ScienCell (2 mentions)
Gelatin coated 100 mm petri dishes, by BD (2 mentions)
Phosphate buffered saline (pbs), by Harvard Bioscience (2 mentions)
Fetal bovine serum (fbs), by ScienCell (2 mentions)
42 protocols using gentamycin
1
Co-culture of Pericytes and Endothelial Cells
2
Isolation and Differentiation of Endothelial Cells from Human Umbilical Cord Blood
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All human UCB samples were collected from donors, who signed an informed consent form, in compliance with Portuguese legislation. The collection was approved by the ethical committees of Dr. Daniel de Matos Maternity Hospital in Coimbra and Hospital Infante D. Pedro in Aveiro. CD34+ cells were isolated from human UCB and differentiated into ECs according to a protocol previously reported by us [6] (link). Briefly, isolated CD34+ cells were cultured in EGM-2 medium (Lonza) supplemented with 20% (v/v) fetal bovine serum (FBS; Life Technologies) and 50 ng/mL of VEGF165 (PeproTech Inc.), on 1% (w/v) gelatin-coated 24-well plates (2×105 cells/well). After 15–20 days ECs are seen in the culture dish. For each experiment, the cells were expanded in 1% (w/v) gelatin-coated 100 mm Petri dishes (BD Falcon) in EGM-2 medium (with all the supplements except FBS and gentamycin/amphotericin) supplemented with 2% (v/v) FBS, 50 µg/mL gentamycin (Biochrom AG) and 1 ng/mL basic fibroblast growth factor (bFGF).
3
Cell Culture and Maintenance Protocol
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HT-29 colon carcinoma cells, MDA-MB-231 breast cancer cells, MCF-7 breast carcinoma cells (all supplied by Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) were maintained in Dulbecco's Modified Eagle Medium (4.5 g/L D-glucose, L-glutamine, pyruvate), which was supplemented with gentamycin (50 mg/L) and fetal bovine serum superior, standardized (Biochrom GmbH, Berlin) (10% v/v), and were passaged once a week. RC-124 healthy human kidney cells (supplied by CLS Cell Lines Service GmbH, Eppelheim, Germany) were maintained in McCoy's 5A (modified, with L-glutamine) medium which was supplemented with gentamycin (50 mg/L) and fetal bovine serum superior, standardized (Biochrom GmbH, Berlin) (10% v/v), and were also passaged once a week. For experiments with RC-124 cells, microtiter plates had been pretreated in the following way: 30 μL of a sterilized gelatine solution (1.5% (m/V)) were added to each well of flat bottom 96-well plates, the plates were covered with their lids, incubated for 1h at 37°C, the excess solution was removed, the wells were washed with PBS 7.4 pH, and the new cell-culture medium was added.
4
Differentiation of THP-1 Macrophages
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The monocytic cell line THP-1 (DSMZ No. ACC 16) was cultured in RPMI 1640 (Biochrom AG, Berlin, Germany) supplemented with 10% FBS superior (Biochrom AG), 2 mM l -glutamine (Biochrom AG), 1 mM Na-pyruvate (Biochrom AG), gentamycin (10 µg/mL) (Biochrom AG), and 1 mM HEPES buffer (Biochrom AG) at 37 °C in a 5% CO2 humidified atmosphere and passaged 2–3 times per week. Cells were used up to passage 20. To perform infection experiments, cells were differentiated into macrophages by stimulation with phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, Munich, Germany) 48 h prior to infection. For that purpose, cells were seeded in a 10 µM PMA solution in RPMI including listed supplements but without antibiotics at a density of 1 × 106 cells per well, applying a 1.5-mL volume together with a six-well cell culture plate (Sarstedt AG, Nümbrecht, Germany). After 24 h of PMA stimulation, the stimulus was removed by washing the adherent cell layer with PBS. Medium including listed supplements but without antibiotics was provided for another 24 h before using THP-1-derived macrophages for infection experiments.
5
Isolation and Expansion of Synovial Mesenchymal Stem Cells
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The assay was approved by the local Clinical Research Ethics Committee and performed in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki). Informed consents were obtained from the patients. The protocol for SMSCs isolation was established as previously described [13 (link)]. Briefly, synovial tissue was collected from the patients with osteoarthritis during the operations. The tissues were cut into small pieces, washed with PBS, and cultured in DMEM medium containing 10% FBS (Invitrogen, U.S.A.), 1% penicillin/streptomycin (Invitrogen), 3% collagense P (Roche, Mannheim, Germany), and 0.5% gentamycin (Biochrom). Tissues were digested at 37°C for 3 h, and then the suppression was centrifuged and seeded in expansion medium DMEM with 10% FBS (Gibco, U.S.A.).
6
Expansion and Maintenance of T Cells
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T cells were kept at a density of ∼106 cells per ml cell culture medium. The T-cell medium consisted of RPMI 1640 (Invitrogen), 1× penincillin/streptomycin (Invitrogen), 5% FCS, 5% human serum, 1 mM sodium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen), 10 mM nonessential amino acids (Invitrogen), 10 mM Hepes (Invitrogen), and 16 μg/ml gentamycin (Biochrom). Human IL-7 and human IL-15 (both PeproTech) were added to the medium to a final concentration of 5 ng/ml each and replenished when fresh culture medium was added to the cells every 2–3 d. The AML cell line ML2 (The CABRI consortium) was retrovirally transduced with genes encoding firefly luciferase (fluc) or HLA-B7. Mycoplasma contamination status was regularly tested.
7
Isolation and Characterization of AD-hMSCs
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Primary AD-hMSCs29 were isolated and identified by immune phenotype and functional characteristics as defined by the International Society for Cellular Therapy5 (link) comprising the presence of CD105, CD73, and CD90, and the absence of CD45, CD34, CD14 or CD11b, CD79α or CD19, and human leukocyte antigen DR isotype (HLA-DR) surface molecules. Cells in passage 2 were cultivated at 37°C in complete medium (minimum essential medium eagle alpha medium; Gibco, Germany), 10% human serum AB (c.c.pro GmbH, Germany), 0.5% gentamycin (Biochrom, Germany) in a T175 culture flask (Sarstedt, Germany) in humidified atmosphere (5% CO2/21% O2). At 80% confluency, AD-hMSCs were harvested through Accutase©-treatment, counted, and DNA transfer was performed.
8
Differentiation of Primary Human Endothelial Cells
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Primary human Endothelial cells (phECs) were differentiated from CD34+ cells isolated from human cord blood as previously described [24 (link)] and were provided in frozen aliquots of 1 million cells at passage 5 by Prof. Gosselet, Université d´Artois, France. After thawing, cells were seeded onto gelatine (0.2% in PBS) coated 10 cm-dishes in ECM-5 medium (ECM from Sciencell with 5 mL of Endothelial cell growth supplement (ECGS), 2.5 mL of Gentamycin 10 mg/mL (BiochromAG, Ref. A-2712) and 25 mL of pre-selected, heat-inactivated FBS; and cultivated at 37 °C, 5% CO2. When cells reached confluency, they were washed three times with PBS, detached with Trypsin/EDTA solution, counted and re-seeded at a concentration of ∼20 000 cells/cm2. Expression of EC marker CD31 was confirmed by flow cytometry and immunofluorescence.
9
Culturing Cancer and Kidney Cells
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A549 lung carcinoma cells and MDA‐MB‐231 breast cancer cells were maintained in Dulbecco's modified Eagle's medium (DMEM; 4.5 g/L d ‐glucose, L‐glutamine, pyruvate), supplemented with fetal bovine serum superior, standardized (Biochrom GmbH, Berlin, 10 % v/v) and gentamycin (50 mg/L) with a weekly passage. RC‐124 healthy human kidney cells were maintained in McCoy's 5 A medium (modified with L‐glutamine) supplemented with fetal bovine serum superior, standardized (Biochrom GmbH, Berlin) and gentamycin (50 mg/L) (10 % v/v), and were also passaged weekly. For experiments with RC‐124 cells, microtiter plates were pretreated as follows: sterilized gelatine solution (1.5 %; 30 μL) was added to each well of a flat‐bottomed 96‐well plate and incubated for 1 h at 37 °C while covered with the lid. The excess solution was removed and the wells were washed with PBS (pH 7.4). The new cell culture medium was immediately added.
10
Culturing Pancreatic and Kidney Cell Lines
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Panc-1, HEK-293 T, BxPC1 and MiaPaCa 2 cell lines were maintained in DMEM and L3.6pl were maintained in RPMI, each supplemented with 10 % (v/v) FCS, and 1 % (v/v) penicillin and streptomycin. Patient derived cell lines 5061, 5072 and 5167 were maintained in RPMI 1640 with Glutamax (Life Technologies) supplemented with 10 % of fetal calf serum (FCS), 200 IU/ml of penicillin-streptomycin, 0.1 mg/ml gentamycin (Biochrom), 50 nmol/ml of human transferrin (Sigma-Aldrich), 0.01 μg/ml of bovine insulin (Sigma-Aldrich), 0.01 μg/ml of recombinant human epidermal growth factor (Pepro Tech), and 0.01 μg/ml of human basic fibroblast growth factor (Pepro Tech).
Cells were cultured at 37 °C in a humidified atmosphere containing 5 % CO2. All cell lines were used at low passage number not exceeding 30 passages.
Cells were cultured at 37 °C in a humidified atmosphere containing 5 % CO2. All cell lines were used at low passage number not exceeding 30 passages.
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