Cell supernatants were collected for evaluation of secreted cytokines. SFs (104) were seeded in 200 μl of complete DMEM in 96-well plates pre-coated with bovine fibronectin (R&D, UK). For siRNA silencing experiments, the first siRNA transfection was performed in 12-well plates and when the first transfection was completed, the cells were detached for a second transfection and re-seeded in 96-well plates. Cells were cultured in the absence or presence of 10 ng/ml IL-1β for 24 hours, then supernatants were collected and frozen. ELISA kits (Dou set, R&D) were used to evaluate the levels of IL-6, MMP3 and CCL2 in the supernatants according to the manufacturer’s instructions. Cell viability was checked using a colorimetric MTS assay (abcam, catalog number ab223881).
Bovine fibronectin
Manufactured by R&D Systems
Sourced in United States
Bovine fibronectin is a high-purity extracellular matrix protein isolated from bovine plasma. Fibronectin is a glycoprotein involved in cell adhesion, growth, migration, and differentiation.
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5 protocols using bovine fibronectin
1
Cytokine Secretion Assay in Fibroblasts
2
Snap-Freezing and Culturing of Artery Samples
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Biopsies were collected in 0.9% NaCl saline solution with penicillin (100 UI/mL) immediately after surgery, macro-sectioned, embedded in Killik (BioOptica, Milan, Italy), and snap-frozen in 2-methyl butane/liquid N2. Since all specimens were surgical waste, to avoid damage or alterations biasing the results, only IMA with surrounding perivascular adipose tissue were included in the study, whilst the fragments from artery skeletonized during grafting procedure were discarded.
Before being snap-frozen, a subset of sample rings from IMA (n = 3) and CPL (n = 6) was kept in culture, as previously described [38 (link)], and 2 rings/fragment were either untreated or submitted to treatment with A740003, added to culture medium at 100 μM for 24 h (t1) or 72 h (t3).
Numbered serial cryosections (10 μm thick) were obtained and dedicated either to confocal videomicroscopy experiments (pair numbers) or to histology and immunofluorescence (odd numbers). For blood flow videomicroscopy experiments, the sections were collected on glass coverslips coated with bovine fibronectin (50 μg/mL in phosphate buffered solution, R&D Systems Inc., Minneapolis, MN, USA).
Reagents, whereas not differently indicated, were purchased from EuroClone S.p.A., Milan Italy and Sigma-Aldrich Saint Louis, MO, USA.
Before being snap-frozen, a subset of sample rings from IMA (n = 3) and CPL (n = 6) was kept in culture, as previously described [38 (link)], and 2 rings/fragment were either untreated or submitted to treatment with A740003, added to culture medium at 100 μM for 24 h (t1) or 72 h (t3).
Numbered serial cryosections (10 μm thick) were obtained and dedicated either to confocal videomicroscopy experiments (pair numbers) or to histology and immunofluorescence (odd numbers). For blood flow videomicroscopy experiments, the sections were collected on glass coverslips coated with bovine fibronectin (50 μg/mL in phosphate buffered solution, R&D Systems Inc., Minneapolis, MN, USA).
Reagents, whereas not differently indicated, were purchased from EuroClone S.p.A., Milan Italy and Sigma-Aldrich Saint Louis, MO, USA.
3
Microglia-Conditioned Medium Effects on Neural Stem Cells
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For monolayer cultures, dishes or wells were pre-coated with l -poly-ornithine (15%, Sigma Aldrich, St. Louis, USA) and bovine fibronectin (2.5 mmol/l, R&D Systems, Minneapolis, Canada). NSCs were grown at 37 °C with 5% CO2. For differentiation, proliferation, and survival experiments, cells from the second or third passage were sown on pre-coated chamber slides or 24-well plates. After 24 h, NSCs were stimulated with conditioned medium of unstimulated microglia (conditioned medium [untreated]), LPS-stimulated microglia (conditioned medium [LPS]), IL4-stimulated microglia (conditioned medium [IL4]), or microglia stimulated with LPS and IL4 (conditioned medium [LPS + IL4]). NSCs stimulated with sheer 10 ng/ml LPS or 50 ng/ml IL4 as well as unstimulated NSCs served as controls. For analyzing migration potential and neurosphere growing, cells were immediately stimulated when seeded.
4
Isolation and Characterization of Fibrocyte-Like Cells
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Human fibrocyte-like cells were isolated according to previously published methods67 (link). Mononuclear cells were isolated from the peripheral blood of healthy volunteers using Ficoll density centrifugation68 (link). The isolated cells were cultured in DMEM supplemented with 20% FBS, penicillin and streptomycin on bovine fibronectin (R&D systems, Minneapolis, MN)-coated 150 mm cell culture dishes (BD Pharmingen, Franklin Lakes, NJ). The medium was changed twice a week. After 7–10 days, the media was aspirated and washed with sterile PBS three times. The adherent cells were determined to be circulating fibrocyte-like cells by a flow cytometric analysis and immunostaining67 (link). Informed consent was obtained from all volunteers, and the protocol was approved by the IRB of Tokushima University Hospital (No. 1586).
5
Conditioned Medium Collection from GSCs
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To collect conditioned medium (CM) from GSCs (X01, 448, 131 and 83), 5 × 105 GSCs (X01, 448, 131 and 83) were plated in bovine fibronectin (R&D System) coated 60 mm dishes, and after 24 h, the medium was replaced with medium with/without additional lentivirus. CM was collected after 48 h of culture, centrifuged at 1000 g for 20 min at 4 °C to remove the debris, and kept in aliquots at −20 °C for further use.
For Recombinant human IGFBP5 protein treatment, 8 × 105 131 or 83 GSCs were plated in 60 mm dishes and incubated overnight. Recombinant human IGFBP5 protein (100 ng/ml; R&D System) was added to the cultures of 131 and 83 cells for 6 h, and harvested cell pellets were used for further study. For IGF1R inhibitor treatment, 8 × 105 83 GSCs were plated in 60 mm dishes and incubated overnight. NVP-AEW541 (10 μM; TargetMol) was added into the culture medium for 3 h, with or without pre-treatment of Recombinant human IGFBP5 protein for 6 h, and harvested cell pellets were used for further study.
For Recombinant human IGFBP5 protein treatment, 8 × 105 131 or 83 GSCs were plated in 60 mm dishes and incubated overnight. Recombinant human IGFBP5 protein (100 ng/ml; R&D System) was added to the cultures of 131 and 83 cells for 6 h, and harvested cell pellets were used for further study. For IGF1R inhibitor treatment, 8 × 105 83 GSCs were plated in 60 mm dishes and incubated overnight. NVP-AEW541 (10 μM; TargetMol) was added into the culture medium for 3 h, with or without pre-treatment of Recombinant human IGFBP5 protein for 6 h, and harvested cell pellets were used for further study.
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