HDFa (American Tissue Culture Collection, Manassas, VA, USA) were cultured in fibroblast basal medium (Lonza, Verviers, Belgium) containing growth supplements (Lonza), such as 2% FBS, 0.1% recombinant human insulin, 0.1% gentamicin sulfate amphotericin B, and 0.1% recombinant human fibroblast growth factor-B. The cells at a density of 5 × 104 cells/mL (200 μL) were seeded in a 96-well plate until 80–85% confluency. The culture supernatant was replaced with 200 μL of serum-free DMEM containing test samples. After 24 h incubation, the cytotoxicity of the test samples was measured using MTT assay, and ELISA was used to determine the level of the wrinkle-forming mediator, MMP-1 (R&D Systems), in the culture supernatant.
Fibroblast basal medium
Manufactured by Lonza
Sourced in United States, Switzerland
Fibroblast basal medium is a cell culture medium formulated to support the growth and maintenance of fibroblast cells. It provides the essential nutrients and growth factors required for fibroblast cell proliferation and survival in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Growth supplement, by Lonza (2 mentions)
Nhlfs, by Lonza (2 mentions)
Mesenchymal stem cell qualified fetal bovine serum, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Mmp 1, by R&D Systems (1 mentions)
Trypan blue, by Sartorius (1 mentions)
B rker chamber, by Sartorius (1 mentions)
Fgm singlequots, by Lonza (1 mentions)
Direct heat co2, by Thermo Fisher Scientific (1 mentions)
Alpha tubulin antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 488 conjugated donkey anti rabbit secondary antibody, by Abcam (1 mentions)
Sb431542, by R&D Systems (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Human primary osteoblasts, by Lonza (1 mentions)
24 well plate, by Corning (1 mentions)
Penicillin streptavidin, by Thermo Fisher Scientific (1 mentions)
Penicillin streptavidin, by Lonza (1 mentions)
2 mercaptoethanol, by Merck Group (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
12 protocols using fibroblast basal medium
1
In Vitro Cytotoxicity and MMP-1 Evaluation
2
Adalimumab Effects on NHDF Cells
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The NHDF cell line (fourth passage) was cultured in the FBM medium (Fibroblast Basal Medium; Lonza, Basel, Switzerland), supplemented with hFGF-B (Human Fibroblast Growth Factor-Basic) and insulin and gentamicin (FGM™ SingleQuots™; Lonza, Basel, Switzerland) at 37°C in a 5% CO2 incubator (Direct Heat CO2; Thermo Scientific, Waltham, MA, USA). The quantity of cells and their viability were monitored by cell counting in the Bürker chamber after staining them with 0.2% trypan blue (Biological Industries, Beit HaEmek, Israel). To test the effects of the drug on gene expression, 8 μg/ml of adalimumab (AbbVie Biotechnology GmbH, Knollstrasse, Germany) was added to the cell culture for 2, 8, and 24 hours. Control cells were not treated with adalimumab. The concentration was customized to the level of adalimumab in the bodies of psoriatic patients.
3
Fibroblast Characterization and Activation
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Fibroblast basal medium, growth supplements, and NHLFs were purchased from Lonza (Walkersville, MD). Human-derived transforming growth factor beta (TGFβ), as well as the TGFβ receptor blocker SB431542, was obtained from R&D Systems, Inc. (Minneapolis, MN). Poly-L-lysine was purchased from Sigma-Aldrich (St. Louis, MO). The anti-Collagen I antibody was purchased from Fitzgerald (Acton, MA), while the alpha tubulin antibody was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX). Alexa-fluor 488-conjugated donkey anti-rabbit secondary antibody, alexa-fluor 488-conjugated alpha-smooth muscle actin (α-SMA) primary antibody, and the beta actin primary antibody were purchased from Abcam (Cambridge, MA). ProLong gold antifade with DAPI mountant was obtained from Molecular Probes (Thermo Fisher Scientific, Waltham, MA).
4
Culturing Primary Human Cell Types
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Primary human fibroblasts (Lonza, Walkersville, MD, CC-2511) were cultured in Fibroblast Basal Medium (Lonza CC-3131) supplemented with human fibroblast growth factor-basic, insulin, gentamicin/amphotericin-B, and 10% fetal bovine serum (Lonza CC-4134). Primary human myoblasts (DV Biologics, Yorba Linda, CA, AM002-F) were cultured in Muscle Cellutions Medium (DV Biologics M-GRO-001-500) supplemented with basal media supplement (DV Biologics M-GRO-0010-S) and 20 ng/ml of human fibroblast growth factor-basic (R&D systems, Minneapolis, MN, 233-FB-025). Primary human osteoblasts (Lonza CCC-2538) were cultured in Osteoblast Basal Medium (Lonza CC-3208) supplemented with 10% fetal bovine serum, ascorbic acid, and gentamicin/amphotericin-B (Lonza CC-4193).
Cells were initially seeded in standard sterile 75-cm2 tissue culture flasks (Corning Life Sciences, Tewksbury, Massachusetts) at a density of 10,000 cells/cm2 and grown to 80% confluency at 37˚C and 5% CO2 (link). Cells were passaged in tissue culture flasks until passage 3, at which time the cells were seeded into 24-well plates (Corning Life Sciences, Corning, NY) at a density of 10,000 cells/cm2 and cultured until 80-90% confluent. CHX solutions were made by diluting 4% chlorhexidine gluconate (Xttrium Laboratories, Mount Prospect, IL) in sterile phosphate buffered saline (PBS, Gibco, Waltham, MA).
Cells were initially seeded in standard sterile 75-cm2 tissue culture flasks (Corning Life Sciences, Tewksbury, Massachusetts) at a density of 10,000 cells/cm2 and grown to 80% confluency at 37˚C and 5% CO2 (link). Cells were passaged in tissue culture flasks until passage 3, at which time the cells were seeded into 24-well plates (Corning Life Sciences, Corning, NY) at a density of 10,000 cells/cm2 and cultured until 80-90% confluent. CHX solutions were made by diluting 4% chlorhexidine gluconate (Xttrium Laboratories, Mount Prospect, IL) in sterile phosphate buffered saline (PBS, Gibco, Waltham, MA).
5
Isolation and Culture of Mouse Embryonic Fibroblasts
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MEFs isolated from 13.5-day embryos were cultured in a 5% CO2 and 3% O2 incubator, in Dulbecco’s modified Eagle’s medium GlutaMAX (Gibco), with 15% fetal bovine serum (Biowest), 100 μM 2-mercaptoethanol (Millipore), 0.01 mM Non-Essential Amino Acids, and penicillin/streptavidin (Gibco) for five or fewer passages, except for 3T3 experiments, performed in a 5% CO2 incubator for seven passages. Cells were treated for 24 hours with 10 μM Nutlin 3a (Sigma-Aldrich) (22 (link)) or 15 μM cisplatin (Sigma-Aldrich). Primary human fibroblasts at low passage (p.2 for TINF2K280E, p.3 for NCI-226-4 and NCI-226-8, and p.4 for TERTP704S) were thawed and cultured in fibroblast basal medium (Lonza) with 20% fetal calf serum, l -glutamin, 10 mM Hepes, penicillin/streptavidin, and gentamicin before quantitative PCR (qPCR) analysis.
6
Isolation and Expansion of hWJCs from Human Umbilical Cords
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hWJCs were isolated from Wharton’s jelly of human umbilical cords according to the protocols approved by the University of Kansas Human Subjects Committee (KU-Lawrence IRB approval #15402) and Stormont-Vail (SMV) Hospital (SMV IRB approval conferred by IRB Chair, Jo-Ann S. Harris, MD). Nursing staff obtained informed consent from patients before collection of the umbilical cords. Three umbilical cords (n = 3) were obtained from Stormont-Vail (Topeka, KS, USA). All cords were from males that were born at full term and delivered under normal delivery conditions.
hWJCs were isolated and cultured according to previous published protocols [23 (link)]. hWJCs were cultured in hWJC medium consisting of 1% penicillin-streptomycin (Life Technologies), 10% fetal bovine serum (FBS) mesenchymal stem cell qualified (MSCq) (Life Technologies), and fibroblast basal medium (Lonza Group Ltd., Basel, Switzerland), and expanded to passage 5 for all experiments.
hWJCs were isolated and cultured according to previous published protocols [23 (link)]. hWJCs were cultured in hWJC medium consisting of 1% penicillin-streptomycin (Life Technologies), 10% fetal bovine serum (FBS) mesenchymal stem cell qualified (MSCq) (Life Technologies), and fibroblast basal medium (Lonza Group Ltd., Basel, Switzerland), and expanded to passage 5 for all experiments.
7
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.
8
Isolation and Expansion of Human Mesenchymal Stem Cells
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Human MSCs were isolated from Wharton’s jelly of umbilical cords according to the protocols approved by the University of Kansas Human Subjects Committee (KU-Lawrence IRB approval #15402) and Stormont-Vail (SMV) Hospital (SMV IRB Approval conferred by IRB Chair, Jo-Ann S. Harris, MD). Informed consent was obtained from the patients before collection of the umbilical cords. Three umbilical cords (n = 3) were obtained from Stormont-Vail (Topeka, KS, United States). All cords were from males that were born at full term and delivered under normal delivery conditions. Human MSCs were isolated and cultured according to previously published protocols (Mellott et al., 2016 (link)). Cells were cultured in medium consisting of 1% penicillin-streptomycin (Life Technologies), 10% fetal bovine serum (FBS) mesenchymal stem cell qualified (MSCq) (Life Technologies), and fibroblast basal medium (Lonza Group Ltd., Basel, Switzerland), and expanded to passage 5 for all experiments. For delivery, cells were harvested, washed in phosphate buffered saline (PBS) and resuspended to a concentration of 1 × 106 cells/ml in sterile PBS.
9
Culturing Alveolar Cells and Lung Fibroblasts
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Human alveolar epithelial cells (A549; Cat#: CCL-185, ATCC, Manassas, VA, USA) were cultured in Dulbecco's Modified Eagle Medium (DMEM) with L-glutamine (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, St Louis, MO, USA), 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies). Normal human lung fibroblasts (NHLFs; Cat#: CC-2512, Lonza, Walkersville, MD, USA) were cultured in fibroblast basal medium (Lonza) supplemented with FGM-2 SingleQuots supplements (Lonza), 100 U/ml penicillin, and 100 µg/ml streptomycin.
10
Cell Culture of Human Lung Fibroblasts and Breast Cancer Cell Lines
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All cell culture supplies were purchased from Thermo Fisher Scientific (Waltham, MA, USA) unless otherwise noted. Human lung fibroblast (HLF) cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA) and cultured in Fibroblast basal medium (FBM) (Lonza, Quakertown, PA, USA) supplemented with 2% fetal bovine serum (FBS), 0.1% insulin, 0.1% r-human fibroblast growth factor (rhFGF) and 0.1% Gentamicin sulfate amphotericin-B (GA-1000) (Lonza, Quakertown, PA, USA). HLFs were used from passages 1 to 10. Human breast cancer cell line MDA-MB-231 was provided by Dr. Shannon Hughes. Lung tropic MDA-MB-231 LM2 and MDA-MB-231 cell lines were cultured in Dulbecco’s modified eagle’s medium (DMEM), supplemented with 1% penicillin-streptomycin, and 10% FBS. BT474 cell line was cultured in Roswell Park Memorial Institute (RPMI) 1640 medium, supplemented with 1% penicillin-streptomycin, and 10% FBS.
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