HCF cells were purchased from ScienCell Research Laboratories (San Diego, CA, USA) and cultured in Fibroblast Medium (ScienCell Research Laboratories) with Fibroblast Growth Supplement to ascertain the cell phenotypes during passages. Cells from passages 3–6 were used for the experiments. The cells were treated with 44 μM ATX for 6 h with or without pretreatment for 30 min with 1 µM HA130, an ATX inhibitor (Santa Cruz Biotechnology, Santa Cruz, CA, USA).
Fibroblast growth supplement
Manufactured by ScienCell
Sourced in United States
Fibroblast growth supplement is a specialized media supplement designed to support the growth and proliferation of fibroblast cells in cell culture systems. It contains a proprietary blend of growth factors, vitamins, and other essential nutrients specifically formulated to optimize the in vitro expansion of fibroblasts.
Automatically generated - may contain errors
Lab products found in correlation
Fibroblast medium, by ScienCell (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Fetal bovine serum (fbs), by ScienCell (4 mentions)
Rpmi 1640 medium, by American Type Culture Collection (2 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin, by ScienCell (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (2 mentions)
Penicillin streptomycin p s, by ScienCell (2 mentions)
Fibroblast medium fm, by ScienCell (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Hcf cells, by ScienCell (1 mentions)
Smooth muscle cell medium, by ScienCell (1 mentions)
Smooth muscle cell growth supplement, by ScienCell (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Hconf, by ScienCell (1 mentions)
Dulbecco s modified eagle s medium dmem f12, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Beas 2b, by Thermo Fisher Scientific (1 mentions)
13 protocols using fibroblast growth supplement
1
ATX Inhibition in Fibroblast Phenotype
2
Culturing Human Lung Cells for Research
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Primary human bronchial fibroblasts (HBFs) and fibroblast medium were purchased from ScienCell (USA). HBFs were cultured in fibroblast medium containing 2% fetal bovine serum (FBS, ScienCell), 1% penicillin‐streptomycin (ScienCell), and 1% fibroblast growth supplement (ScienCell). Cells at passages 4–8 were used for the experiments. Human lung fibroblast 1 (HFL1, ATCC CCL‐153) cells and F‐12K (Kaighn's modification of Ham's F‐12 medium, ATCC 30‐2004) were purchased from ATCC (Manassas, VA, USA). HFL1 were cultured in F‐12K containing 10% FBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin‐streptomycin (Invitrogen, Carlsbad, CA, USA). Primary human bronchial smooth muscle cells (HBSMCs) and smooth muscle cell medium were purchased from ScienCell (Carlsbad, CA, USA). HBSMCs were cultured in smooth muscle cell medium containing 2% FBS (ScienCell), 1% penicillin‐streptomycin (ScienCell), and 1% smooth muscle cell growth supplement (ScienCell). BEAS‐2B (ATCC CRL‐9609) cells and RPMI‐1640 Medium (ATCC 30‐2001) were purchased from ATCC. HFL1 were cultured in RPMI‐1640 Medium containing 10% FBS (Gibco, USA) and 1% penicillin‐streptomycin (Invitrogen, USA). Above primary cells or cell lines were all cultured and incubated at 37°C under a humidified atmosphere containing 5% CO2.
3
Cardiac Fibroblast Culturing and Substrate Stiffness
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Commercially available primary hCF isolated from right ventricles of human male donors (age 39–42 years) (CC-2904, Lonza, Walkersville, MD, USA) was expanded in complete FGM-3 medium (Lonza) to passage P3 and routinely cultured between passages P3-P5 in medium consisting of 45% Dulbecco’s modified Eagle’s medium (DMEM), 45% F12 (Gibco Invitrogen, Burlington, ON, Canada), 10% fetal calf serum (Invitrogen), 1000 U/mL penicillin/streptomycin (Invitrogen), and fibroblast growth supplement (ScienCell, Carlsbad, CA, USA). Rat embryonic fibroblasts (REF) and human MRC-5 cells were used for selected experiments and cultured in Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal calf serum (Gibco), 1000 U/mL penicillin/streptomycin (Invitrogen). Soft silicone culture substrates (ExCellness Biotech, Lausanne, Switzerland) were used to mimic the elastic range of healthy and diseased tissue and to control myofibroblast activation. We selected 3 kPa soft, 26 kPa medium-stiff, and 65 kPa stiff substrates to model increasing severity of fibrotic scar stiffening [38 (link)]. Silicone culture substrates were oxygen plasma-treated and coated with 2 μg/cm2 fibronectin (Millipore, Billerica, MA, USA) to promote cell attachment [39 (link)].
4
Isolation and Cultivation of Human Conjunctival Fibroblasts
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HConFs from ScienCell Research Laboratories (San Diego, CA, USA) were isolated from the human conjunctiva. HConFs are characterized by a spindle-shaped morphology and positivity for immunofluorescence staining with anti-fibronectin antibodies. HConFs were maintained and cultured at 37°C in a humidified incubator with 5% CO2 in fibroblast medium (ScienCell Research Laboratories) containing fibroblast growth supplement (undisclosed formulation), 2% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin.
5
Isolation and Culture of Human Conjunctival Fibroblasts
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Primary human conjunctival fibroblasts (HConFs) were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA). Following the manufacturer’s instructions, HConFs were isolated from the conjunctiva of human eyes, dissected, and digested by enzymes. HConFs cultures were grown in fibroblast medium (ScienCell) supplemented with 5% fetal bovine serum (ScienCell), fibroblast growth supplement (ScienCell), and penicillin–streptomycin (100 U/mL and 100 mg/mL) solution (ScienCell). Primary HConFs were generated using an expansion culture of human Tenon’s explants propagated in Dulbecco’s modified Eagle’s medium (DMEM–F12; Gibco, Carlsbad, CA, USA) supplemented with 10% FBS (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich, St. Louis, MO, USA). Cells were maintained in the logarithmic growth phase. HConFs at passages 3 to 6 were used for all experiments. Experiments were replicated six times.
6
Primary Cell Culturing for Lung Research
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Primary HBFs and fibroblast medium (FM) were obtained from ScienCell (USA). HBFs at passages 4−8 were cultured in fibroblast medium containing 2% foetal bovine serum (FBS; ScienCell) supplemented with 1% penicillin‐streptomycin (P/S; ScienCell) and 1% fibroblast growth supplement (ScienCell). Human lung fibroblast 1 (HFL1, ATCC CCL‐153) cells, F‐12K (Kaighn's modification of Ham's F‐12 medium, ATCC 30−2004), BEAS‐2B (ATCC CRL‐9609) cells, A549 (ATCC CCL‐185) and RPMI‐1640 medium (ATCC 30−2001) were obtained from ATCC (Manassas, USA). HFL1 cells were cultured in F‐12K medium containing 10% FBS (Gibco, USA) and 1% P/S (Invitrogen, USA), whereas BEAS‐2B and A549 cells were cultured in RPMI‐1640 medium containing 10% FBS and 1% P/S. The above primary cells or cell lines were all incubated at 37°C under a humidified atmosphere containing 5% CO2.
7
Establishing DM-OA Synovial Fibroblast Model
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To establish DM‐OA cell model in vitro, rat synovial fibroblasts were purchased from iCell Bioscience Inc. (Shanghai, China) and maintained in DMEM medium (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 1% fibroblast growth supplement (Sciencell, Carlsbad, CA, USA) and 10% fetal bovine serum (Sigma‐Aldrich, St Louis, MO, USA). Cells were infected with lentivirus particles to overexpress PSTPIP2. Two days after lentivirus infection, cells were added with normal glucose (5.5 mmol/L, Aladdin regents, Shanghai, China), high glucose (25 mmol/L), IL‐1β (5 ng/mL, MedChemExpress, Monmouth Junction, NJ, USA) or ERK inhibitor U0126 (10 μM, MedChemExpress) according to the group information. Two days later, cells were collected for further testing.
8
Culturing Human Pulmonary Microvascular ECs and Fibroblasts
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First passage human pulmonary microvascular endothelial cells (ECs) and human dermal fibroblasts (HDF) were obtained from ScienCell Research Laboratories (San Diego, California, USA), and the cells were cultured in an endothelial cell medium (ECM; 5%FBS, 1% endothelial cell growth supplement, and 1% penicillin/streptomycin solution) and fibroblast medium (FM; 5%FBS, 1% fibroblast growth supplement, and 1% penicillin/streptomycin solution), respectively. All mediums were purchased from ScienCell Research Laboratories (San Diego, California, USA). Cells were cultured at 37°C in the incubator with an atmosphere of 5% CO2 air. Every 3 to 5 days, cells were passaged when they reached 70-80% confluency.
9
Endometrial Fibroblast Isolation and Culture
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Endometrial samples were obtained from 5 young women (24-32 years old) undergoing oocyte donation. The biopsies were collected after each patient in accordance with the requirements of the Ethical Committee of Cruces Hospital signed informed consent. Endometrial tissue was rinsed with phosphate-buffered saline (PBS) supplemented with antibiotics and minced into 1-2 mm 3 pieces. The fragments were digested with 0.3% collagenase II (Gibco Life Technologies, Gaithersburg, MD, USA) in fibroblast medium (FM, ScienCell Research Laboratories, San Diego, CA, USA) with antibiotics at 37 8C. The resulting cell suspension was seeded into culture flasks and maintained with FM supplemented with Fibroblast Growth Supplement, 2% fetal bovine serum (FBS) and antibiotics (penicillin/streptomycin) (termed complete FM) (ScienCell Research Laboratories, San Diego, CA, USA) in a humidified atmosphere at 37 8C with 5% CO 2 . Cell viability was assessed by trypan blue dye exclusion. Passage 3-4 cells were used in all experiments. The fibroblast-like morphology of cells isolated was checked by phase contrast microscopy.
10
Fibroblast Extracellular Matrix Regulation
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High-glucose (25 mM) Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco-BRL (Rockville, MD, USA). Fibroblast growth supplement (FGS) was purchased from ScienCell Research Laboratories (Carlsbad, CA, USA). Antibiotic solutions (penicillin/streptomycin) were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The Sirius Red/Fast Green Collagen Staining Kit was purchased from Chondrex (Woodinville, WA, USA). The NucleoSpin RNA Kit, RNA PCR Kit (Prime Script), and TB Green® Premix Ex Taq™ (Tli RNaseH Plus) solution were purchased from Takara Bio (Otsu, Japan). Enzyme-linked immunosorbent assay (ELISA; col1a1 and col3a1, FN, and elastin) kits were purchased from MyBioSource (San Diego, CA, USA).
Monoclonal IgG primary antibodies, including mouse anti-FAK, -phospho-FAK, and -GAPDH, were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Appropriate biotin-conjugated goat anti-mouse IgG secondary antibodies were purchased from Abcam (Cambridge, UK). Small interfering ON-TARGETplus Human PTK2 siRNA and Dharma FECT 1 Transfection Reagent were purchased from Dharmacon (Lafayette, CO, USA).
The Sirius Red/Fast Green Collagen Staining Kit was purchased from Chondrex (Woodinville, WA, USA). The NucleoSpin RNA Kit, RNA PCR Kit (Prime Script), and TB Green® Premix Ex Taq™ (Tli RNaseH Plus) solution were purchased from Takara Bio (Otsu, Japan). Enzyme-linked immunosorbent assay (ELISA; col1a1 and col3a1, FN, and elastin) kits were purchased from MyBioSource (San Diego, CA, USA).
Monoclonal IgG primary antibodies, including mouse anti-FAK, -phospho-FAK, and -GAPDH, were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Appropriate biotin-conjugated goat anti-mouse IgG secondary antibodies were purchased from Abcam (Cambridge, UK). Small interfering ON-TARGETplus Human PTK2 siRNA and Dharma FECT 1 Transfection Reagent were purchased from Dharmacon (Lafayette, CO, USA).
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