Four types of human colon carcinoma cell lines (HT-29, WiDr, CaCo-2, and Colo320) were obtained from American Type Culture Collection (Rockville, MD, USA). HT-29 was incubated in McCoy’s supplemented with 10% fetal bovine serum (FBS). WiDr and CaCo-2 were cultured in minimum essential medium Eagle (Sigma Chemical Co., St. Louis, MO, USA) with 10% FBS and high glucose. Colo320 was maintained in RPMI-1640 medium (Sigma Chemical Co.) supplemented with 10% FBS. HUVEC was purchased from Kurabo Co. (Osaka, Japan). HUVECs were maintained in HuMedia-EG2 medium supplemented with 2% FBS, 5 ng/mL basic Fibroblast growth factor, 10 µg/mL heparin, 10 ng/mL epidermal growth factor, and 1 µg/mL of hydrocortisone according to the supplier’s instruction (Kurubo Co.). Fibroblast was obtained from Lonza (Walkersville, MD, USA) and cultured in Fibroblast medium-2 (FBM-2) medium supplemented with 2% FBS, 1 ng/mL basic Fibroblast growth factor, and 1 mg/mL insulin, according to the supplier’s instructions. All the cells were cultured at 37°C in a humidified atmosphere of 5% CO2 in air.
Basic fibroblast growth factor (bfgf)
Manufactured by Lonza
Sourced in United States
BFGF is a laboratory reagent used to support the growth and proliferation of cells in cell culture systems. It functions as a growth factor, providing essential nutrients and signaling molecules to promote cell division and survival. The core function of BFGF is to maintain and expand cell populations in vitro.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Lonza (5 mentions)
Hydrocortisone, by Lonza (4 mentions)
Ascorbic acid, by Lonza (4 mentions)
Ga 1000, by Lonza (4 mentions)
Vascular endothelial growth factor (vegf), by Lonza (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions)
Epidermal growth factor (egf), by Lonza (3 mentions)
Caco 2, by Merck Group (2 mentions)
Human umbilical vein endothelial cells (huvec), by Kurabo (2 mentions)
Rpmi 1640 medium, by Merck Group (2 mentions)
Colo320, by Merck Group (2 mentions)
Heparin, by Lonza (2 mentions)
Insulin like growth factor 1, by Lonza (2 mentions)
Amphotericin b, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
L glutamine, by Lonza (2 mentions)
Trypsin edta, by Lonza (2 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (2 mentions)
Dmem f12, by Thermo Fisher Scientific (2 mentions)
Caco 2, by American Type Culture Collection (1 mentions)
15 protocols using basic fibroblast growth factor (bfgf)
1
Culturing Colorectal Cancer and Endothelial Cells
2
Culturing Colorectal Cancer Cell Lines
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Four cell lines derived from human colorectal carcinoma were examined: HT-29, WiDr, CaCo-2 and Colo320. All cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). The HT-29 was cultured in McCoy’s supplemented with 10% fetal bovine serum (FBS). WiDr and CaCo-2 were maintained in minimum essential medium eagle (Sigma Chemical Co., St. Louis, MO, USA) with high glucose and 10% FBS. Colo320 was maintained in RPMI-1640 medium (Sigma Chemical Co.) supplemented with 10% FBS. Fibroblasts were obtained from Lonza Walkersville Inc. (Walkersville, MD) and maintained in FBM-2 medium supplemented with 2% FBS, 1 ng/ml bFGF, and 1 mg/ml insulin. Human umbilical vein endothelial cell (HUVEC) was obtained from Kurabo Co. (Osaka, Japan). HUVEC were culture in HuMedia-EB2 medium supplemented with 2% FBS, 5 ng/ml of basic fibroblast growth factor, 10 µg/ml heparin, 10 ng/ml epidermal growth factor, and 1 µg/ml hydrocortisone according to the supplier’s instructions (Kurabo Co.). Fibroblast was obtained from Lonza (Walkersville, MD) and maintained in FBM-2 medium supplemented with 2% FBS, 1 ng/mL basic fibroblast growth factor, and 1 µg/ml insulin according to the supplier’s instructions. All cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air.
3
Endothelial Cell Culture and Characterization
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Pooled HUVEC (from 3 to 6 individual donors, Lonza, Cologne, Germany) and HUAEC (from 250 individual donors, PeloBiotech GmbH, Planegg, Germany) were cultivated in EGM-2 Bullet Kit containing endothelial cell culture medium supplemented with 2% (v/v) fetal bovine serum, vascular endothelial growth factor, basic fibroblast growth factor, human epithelial growth factor, insulin-like growth factor-1, hydrocortisone, heparin, ascorbic acid, gentamycin, and amphotericin B (Lonza) in a standard humidified incubator at 37 °C with 5% (v/v) CO2. Cells of passage 5 were seeded in 4-well PCA chamber slides (Sarstedt, Nürnbrecht, Germany) at a cell density of 15,000 cells/well with 1 mL medium per well, and cultivated for up to 7 days. Medium was exchanged at day 2 and day 4 of cultivation.
4
Isolation and Culture of Rabbit Bone Marrow MSCs
5
Endothelial Cell Differentiation Induction
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HRMEC were purchased from Cell Systems (Kirkland, WA) and cultured as described previously (Nawaz et al., 2013) in endothelial basal medium-2 (EBM-2) enriched with endothelial growth medium-2 MV Bulletkit, containing, besides fetal calf serum [FCS; final concentration 5% (v/v)], the growth factors epidermal growth factor, vascular endothelial growth factor (VEGF), basic fibroblast growth factor and insulin-like growth factor 1 (Lonza, Verviers, Belgium). Three days after seeding in 6-well plates, differentiation of HRMEC was induced by addition of human recombinant IL-1, human recombinant TNF-, human recombinant TGF-1 or human recombinant connective tissue growth factor (CTGF) (all from PeproTech, Rocky Hill, NJ) or combinations thereof and replacement of the growth-factor enriched endothelial cell growth medium by EBM-2 supplemented with 3% FCS. As a control to preserve the endothelial cell type, cultures in one well of each plate were contained in the endothelial cell growth medium supplemented with 10 ng/ml of VEGF. The differentiation medium was replaced every two days. After 4 days of stimulation, cell morphology and gene expression were investigated. Changes in cell morphology were documented using an Axiovert 200M inverted microscope, equipped with an EC Plan-Neofluar 10× phase contrast objective (Carl Zeiss, Jena, Germany).
6
Endothelial Cell Inflammation Modeling
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Human aortic endothelial cell line (HAEC) (Lonza), a human aortic endothelial primary cell obtained at passage 2. These cells are very well characterised and were cultured in Endothelial Cell Basal Medium-two and the following growth supplements: hydrocortisone, basic fibroblast growth factor, vascular endothelial growth factor, insulin-like growth factor, ascorbic acid, heparin, FBS, human epidermal growth factor, gentamicin, amphotericin B (Lonza). Experiments were performed at passage 4 where native LDL and Mox-LDL (100 mg/ml), IL-18 (250 pg/ml) and TNF-a (10 ng/ml), sterilised by filtration through a 0.22-mm membrane (Millipore), were added to the culture medium and incubated with the endothelial cells for 24 hours at 37 C under 5% CO 2 . The experiments were performed in triplicate.
7
Hemangiosphere Formation Assay
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To induce hemangiospheres formation, HB cells were grown in suspension in phenol red free DMEM:F-12 medium (GIBCO) supplemented with GlutaMAX (GIBCO), B27 Supplement 50X (GIBCO), 20 ng/ml EGF (Lonza, Basel, Switzerland), 20 ng/ml b FGF (Lonza), and 1% penicillin/streptomycin (GIBCO). 5 × 104 cells/ml were plated at a density of on ultra-low attachment 75 cm2 cell culture flasks (Corning, Corning, NY, US). Cultures were treated or not with 100 μM Propranolol or ICI for 7 days.
8
Sodium Iodate-Induced Retinal Pigment Epithelial Cell Signaling
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Sodium Iodate (NaIO3; 71702) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Retinal Pigment Epithelial Cell Growth Medium (RtEGMTM; #00195407) with supplements including 2% FBS, 2% L-glutamine, 0.5% bFGF, 0.1% GA-1000 was purchased from LONZA (Walkersville, MD, USA). Dulbecco’s modified Eagle’s medium (DMEM; 12800-017), fetal bovine serum (FBS; 26140-079), penicillin/streptomycin (10378-016), and 0.25% trypsin (25200-072) and other cell culture reagents were purchased from Gibco (Gaithersburg, MD, USA). Primary antibodies including phosphor-Akt (ser473; #4058, Thr308; #9275), total Akt (#9272), phospho-ERK (#4370), ERK (#4695), phosphor-JNK (#9251), JNK (#9252), phosphor-p38 (#4511), p38 (#9212), phosphor-IκBα (#9246), and IκBα (#9242) (Cell Signaling Technology, Inc., Danvers, MA, USA), and β-actin polyclonal antibody (SC-47778) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) were used for western blotting analysis. Human pentraxin 3 (PTX3; DY1826) ELISA kit was purchased from R&D System, Inc. (Minneapolis, MN, USA). U0126 (BML-EI282), LY194002 (BML-ST420), SP600125 (BML-EI305), SB203580 (BML-EI286), BAY 11-7082 (BML-EI278) (Enzo Life Sciences, Farmingdale, NY, USA), and NAC (A7250) (Sigma-Aldrich, St. Louis, MO, USA) were used for inhibitor reagents.
9
Isolation and Culture of Rat Bone Marrow Stem Cells
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Tibias and femurs of rats were cut at the epiphyses and perfused with alpha minimal essential medium (αMEM) medium (Sigma-Aldrich, St. Louis, MO) supplemented with 15% fetal bovine serum (FBS) (Lonza Walkersville, Inc., USA). After centrifugation and washing with αMEM medium, bone marrow stem cells were filtered and plated in 25 cm2 flasks (Corning Life Sciences – ALP, Chorges, France) with αMEM + 15% FBS and 1 ng/mL of basic fibroblast growth factor (bFGF) (PeproTech EC Ltd, London, UK). Fresh αMEM medium with 10% FBS and bFGF was added after 48h and successively changed 2 to 3 times per week. After reaching 85% to 90% of confluence, cells were collected using Trypsin-EDTA (Lonza Walkersville, Inc., USA) and seeded in 6-well plates at 13000 cells/cm2 in αMEM + bFGF. Treatments were started as described below when cells reached confluence. Experiments were performed three times.
10
Cultivating Human Microvascular Endothelial Cells
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Human Dermal Microvessels Endothelial Cells (HDMEC) were purchased from Promocell and grown in Endothelial Cell Medium MV (Promocell) containing 5% Fetal Calf Serum (FCS) and endothelial cell growth supplements at 37°C/5%CO2. Human Cerebral Microvessels Endothelial Cells (hCMEC/D3) were a gift from P.O. Couraud at Institut Cochin, Paris, France, and were grown in Endothelial Cell Basal Medium-2 (Lonza) supplemented with 5% of FCS, 1.4 μM hydrocortisone (Lonza), 5 μg/ml ascorbic acid (Lonza), 1 ng/ml b-FGF (Lonza), at 37°C in 5% CO2.
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