Human umbilical vein endothelial cells (HUVECs) and normal human lung fibroblasts (NHLF) were purchased from Lonza (Walkersville, MD) and used before passage 6 and passage 8 respectively. HUVECs were cultured in EGM-2 media containing 2% FBS and supplements such as hEGF, hydrocortisone, VEGF, hFGF-B, R3-IGF-1, ascorbic acid, heparin, and gentamicin/amphotericin-B (Lonza, Walkersville, MD) [44 (link)]. NHLFs were cultured in FGM-2 media containing 2% FBS and supplements such as hFGF-B, insulin, and gentamicin/amphotericin-B (Lonza, Walkersville, MD) [45 (link)]. U87-MG cells, a gift from Professor Nathan D. Price (ISB, Seattle), were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin [29 (link)]. All cell types were cultured at 37 °C in 5% CO 2.
Gentamicin amphotericin b
Manufactured by Lonza
Sourced in United States, Switzerland
Gentamicin/amphotericin-B is a laboratory product that serves as an antibiotic and antifungal agent. It is commonly used in cell culture applications to prevent bacterial and fungal contamination.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Lonza (9 mentions)
Human epidermal growth factor (hegf), by Lonza (8 mentions)
Hydrocortisone, by Lonza (8 mentions)
Ascorbic acid, by Lonza (8 mentions)
Heparin, by Lonza (5 mentions)
L glutamine, by Lonza (5 mentions)
Insulin, by Lonza (4 mentions)
Dexamethasone (dex), by Lonza (4 mentions)
Vascular endothelial growth factor (vegf), by Lonza (3 mentions)
Huvecs, by Lonza (3 mentions)
R3 igf 1, by Lonza (3 mentions)
Ebm 2 medium, by Lonza (3 mentions)
Bovine brain extract, by Lonza (3 mentions)
Epidermal growth factor (egf), by Lonza (3 mentions)
Hfgf b, by Lonza (2 mentions)
Penicillin streptomycin, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Human umbilical vein endothelial cells huvecs, by Lonza (2 mentions)
Fibronectin, by Merck Group (2 mentions)
Type 1 collagen, by BD (2 mentions)
36 protocols using gentamicin amphotericin b
1
Culture and Maintenance of HUVEC, NHLF, and U87-MG Cells
2
Fetoplacental Villous Endothelial Cell Isolation and Culture
3
Culturing Normal Mammary Epithelial Cells
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HMLER and HMLER-shEcad cells derived from normal mammary epithelial cells, were gifted to us by Prof. R. A. Weinberg (Whitehead Institute, MIT). The cells were cultured using Mammary Epithelial Cell Growth Medium (MEGM) containing BPE, hydrocortisone, hEGF, insulin, and gentamicin/amphotericin-B (Lonza). The cells were handled in a sterile environment at all times and cultured in an incubator that was maintained at 37 °C, with an internal atmosphere containing 5% CO2.
4
Cultivation of Human Lung Microvascular Endothelial Cells
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HLMVECs were obtained from small vessels within normal lung tissue (Lonza, Walkersville, MD, USA, http://www.lonza.com /). HLMVECs were cultured in microvascular endothelial growth medium (EBM‐2 Basal Medium supplemented with human Epidermal Growth Factor, Vascular Endothelial Growth Factor, R3‐Insulin‐like Growth factor‐1, Ascorbic Acid, Hydrocortisone, human Fibroblast Growth Factor‐Beta, 5% Fetal bovine Serum, Gentamicin/Amphotericin‐B and 1% penicillin/streptomycin [Lonza, Walkersville, MD, USA]) and incubated in a humidified incubator with 5% CO2 at 37°C. The growth medium was changed 24 hours after seeding and every other day thereafter. Cells were cultured when they were 70%–85% confluent. HLMVECs with the total passage number <9 were used in all the experiments. Although 5% FBS itself contains extracellular vesicles, we chose not to remove the serum from the culture medium due to excessive cell death of HLMVECs with serum starvation.
5
Cell Culture Conditions for Melanoma and Fibroblast
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The B16F10 murine melanoma cells (ATCC, Manassas, VA, USA), CCD-986sk cells (KCLB, Seoul, Korea), and NF-κB luciferase reporter NIH-3T3 stable cells (Panomics, Fremont, CA, USA) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% fetal bovine serum (FBS), 100 units/mL of penicillin, and 100 μg/mL of streptomycin in culture flasks in a CO2 incubator with a humidified atmosphere of 5% CO2 in air at 37 °C. The Normal Human Dermal Fibroblast cells (NHDF, CELLnTEC, Bern, Switzer-land) were cultured in Fibroblast Basal Medium (Lonza, Hayward, CA, USA), supplemented with FGM-2 singleQuots (hGFG, insulin, FBS and gentamicin/amphotericin-B; Lonza, Hayward, CA, USA) in culture flasks in a CO2 incubator with a humidified atmosphere of 5% CO2 in air at 37 °C.
6
Murine and Human Cell Culture Protocols
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The murine dendritic cell line, JAWS II, was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). JAWS II was cultured in the presence of alpha-minimum essential media (Lonza, Basel, Switzerland) complemented with 20% fetal bovine serum (FBS, Life Technologies; Grand Island, NY, USA), 1% penicillin-streptomycin (Sigma-Aldrich, St. Quentin Fallavier, France), 4 mM L-glutamine (Lonza), 1 mM sodium pyruvate (Lonza), and 5 ng/mL of Granulocyte-Macrophage Colony Stimulating Factor (Miltenyi Biotec; San Diego, CA, USA). The cells were maintained in culture in a humified incubator at 37 °C and 5% CO2.
The MDA-MB-231 human breast cancer cells were purchased from ATCC and cultured at 5% CO2 at 37 °C in Dulbecco’s modified Eagle media (Lonza), supplemented with 10% FBS, 2 mM glutamine, 10% penicillin-streptomycin.
Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and grown at 37 °C and 5% CO2 in endothelial basal cell growth medium, supplemented with 2% FBS, 1% streptomycin-penicillin, 0.1% ascorbic acid, 0.1% human epidermal growth factor, 0.1% heparin, 0.1% VEGF, 0.1% gentamicin-amphotericin B, 0.04% hydrocortisone, 0.4% human bFGF, and 0.1% R3-IGF-1 (all from Lonza).
The MDA-MB-231 human breast cancer cells were purchased from ATCC and cultured at 5% CO2 at 37 °C in Dulbecco’s modified Eagle media (Lonza), supplemented with 10% FBS, 2 mM glutamine, 10% penicillin-streptomycin.
Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza and grown at 37 °C and 5% CO2 in endothelial basal cell growth medium, supplemented with 2% FBS, 1% streptomycin-penicillin, 0.1% ascorbic acid, 0.1% human epidermal growth factor, 0.1% heparin, 0.1% VEGF, 0.1% gentamicin-amphotericin B, 0.04% hydrocortisone, 0.4% human bFGF, and 0.1% R3-IGF-1 (all from Lonza).
7
Senescence and TRF in HSMM
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Human skeletal muscle myoblasts (HSMM) were purchased from Lonza (Walkersville, MD, USA). The cells were cultured in Skeletal Muscle Basal Medium (SkBM) supplemented with human epidermal growth factor, fetal bovine serum, dexamethasone, L-glutamine and gentamicin/amphotericin B (Lonza, Walkersville, MD USA). Cells were cultivated at 37°C in a humid atmosphere containing 5% CO2. After that, the myoblasts were expanded extensively to reach replicative senescence, which was manifested by the decline in proliferative capacity and accumulation of senescence biomarker in culture [15 (link)]. For each passage, the population doubling (PD) of cells was calculated as: ln(N/n)/In2, where N is the number of cells at harvest stage and n is the number of cells at seeding stage. In this study, the cells were divided into 4 groups, which were young control (PD < 15; without treatment), young TRF (PD < 15; with TRF treatment), senescent control (PD > 20; without treatment) and senescent TRF (PD > 20; with TRF treatment).
8
Hypoxic Conditioning of HUVECs
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HUVECs used in this study were either obtained from the maternity ward of Kuopio University Hospital by the approval of the Kuopio University Hospital Ethics committee and used at passage 6 or purchased from Life Technologies and used at passage 8. HUVECs were maintained in endothelial cell growth medium (EGM; 0.1% human epidermal growth factor, 0.1% hydrocortisone, 0.1% Gentamicin-Amphotericin-B, 0.4% bovine brain extract, 2% FBS; Lonza) on cell culture flasks coated with 10 g/ml fibronectin (Sigma, St Louis, MO, USA) and 0.05% gelatin in phosphate-buffered saline. To induce hypoxia, cells were incubated in Ruskinn Invivo2 400 hypoxia workstation (The Baker Company) in the presence of 1% O2 and 5% CO2 for 8 h.
9
Leiomyoma Cell Lines: Rat ELT-3 and Human huLM
10
Human Skeletal Muscle Myoblast Lifespan
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Human Skeletal Muscle Myoblasts (HSMM) from 20 years old female Caucasian donor were purchased from Lonza (Walkersville, MD, USA). The skeletal muscle myoblasts were cultured in Skeletal Muscle Basal Medium (SkBM) with fetal bovine serum (FBS), L-glutamine, human epidermal growth factor (hEGF), dexamethasone, and gentamicin/amphotericin-B as supplements to the media (Lonza, Walkersville, MD, USA). Cells were cultivated at 37°C with 5% CO2 atmosphere. The skeletal muscle myoblast cell then underwent serial passaging until it reached cellular senescence. The population doubling (PD) of the cell was calculated for each passage according to the formula ln (N/n)/ln 2 as N is the number of cells at harvest stage and n is the number of cells at seeding stage [33 (link)]. The starting PD for this research was PD 8. The skeletal muscle myoblast cells reached cellular senescence when the cells were unable to proliferate in culture, even with consecutive replenishment. Myoblast cells were considered young at PD 14 and senescent at PD 21.
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