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Human fibronectin

Manufactured by Roche
Sourced in Switzerland, Germany, Sweden

Human fibronectin is a high-molecular-weight glycoprotein found in the extracellular matrix and plasma. It plays a crucial role in cell adhesion, migration, and differentiation. This lab equipment product provides a reliable source of human fibronectin for various research applications.

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11 protocols using human fibronectin

1

Culturing Enteric Nervous System Cells

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Dissociated cells from the muscle layers, including ENS cells, were grown on tissue culture plates (Fisher Scientific, Roskilde, Denmark) pretreated with 20 μg/mL human fibronectin (Roche, Switzerland). To maximize spontaneous aggregation, fibronectin was reduced or omitted in some cultures. Cells were grown in a 1:1 mixture of Dulbecco’s modified Eagle medium (Thermo Fisher Scientific) and Ham’s F-12 (GIBCO/Invitrogen) with l-glutamine, B27 and N2 (GIBCO/Invitrogen), 10 ng/mL human recombinant fibroblast growth factor 2 (basic) (FGF2) and 10 ng/mL human epidermal growth factor (both R&D Systems, Minneapolis, MN), and 10 U/mL penicillin, 100 μg/mL streptomycin (GIBCO/Invitrogen), 50 μg/mL gentamicin (Sigma-Aldrich Australia), and 50 μg/mL metronidazole (Sigma-Aldrich Australia), this is referred to as nTCM. In some cases nTCM was supplemented with 3 μM glycogen synthase kinase 3 (GSK3) inhibitor CHIR-99021 (6-[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)pyrimidin-2-yl]amino]ethylamino]pyridine-3-carbonitrile; Tocris Bioscience/R&D Systems, Minneapolis, MN), which simulates Wnt signaling.25 (link)
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2

Modulating p53 in RASM Cells

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Glass coverslips (Fisher Scientific) were coated with 5 µg/mL human fibronectin (Roche Applied Science) and placed in 24 well plates for two hours. RASM cells in DMEM 10%BGS 1%P/S were plated at a density of 10 000 cells/well. After 24 hours of incubation, the media on the cells was switched to serum free media and the cells were incubated for an additional 24 hours. The p53 inhibitor, pifithrin α (PFA) (20 µM) (Sigma-Aldrich) or the genotoxic drug doxorubicin (Adriamycin) (500 ng/mL) (Sigma-Aldrich) was added to the wells and incubated for 24 hours [26] (link).
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3

Lipid Droplet Dynamics in U2OS Cells

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U2OS cells were seeded in LabTEK chamber slides coated with 5 μg human fibronectin (Roche Diagnostics). Cells were treated with 200– 400 μM oleic acid for 8 h or overnight. Cells were imaged in fresh culture medium or culture medium containing oleic acid including 10 mM HEPES pH 7.3. LDs were stained either with LipidTOX green or LipidTOX deep red. Live cell microscopy was performed with a Leica TCS SP8 confocal microscope or a LSM880 confocal microscope with an Airyscan module. Live cell microscopy for fluorescently tagged NMIIa at LDs was performed in nine independent experiments; GFP-FMNL1 at LDs in four independent experiments; GFP-FMNL1 with BFP-Lifeact at LDs in six independent experiments and BFP-Lifeact/BFP-actin at LDs in four independent experiments. LD dynamics with blebbistatin was assessed in four independent experiments and with cytochalasin D in one experiment.
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4

Microcontact Printing of Fibronectin Patterns

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We followed the protocol by Théry et al for preparation of microcontact-printed substrates with minor modifications [40 ]. We created a silicon master containing disk structures using deep reactive ion etching. From this master, polydimethylsiloxan (PDMS, Sylgard 184 silicone elastomere) negatives were cast that served as stamps for microcontact printing.
We used a Kinpen 11 (Neoplas Control) for activation of glass substrates under sterile working conditions. The glass substrates were then stamped with human fibronectin (Roche) that was allowed to adhere to PDMS stamps before. After this the substrates were treated with PLL(20)-g-[3 (link), 5 (link)]-PEG(2) (SuSoS Surface Technology) to prevent cell adhesion outside of fibronectin-printed areas. We used disks with an area of 3000 (µm)2.
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5

Characterization of B16F1 GFP Melanoma Cells

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B16F1 GFP cells [18 (link)] carry deletions of exons 1α, 1β, and 2 of the Ink4a/Arf gene locus, resulting in loss of p16Ink4a and p19Arf protein expression and altered p53 protein expression without detectable gene mutations, however with no activating BRAF mutations in exons 11 and 15 [19 (link)]. B16F1 cells were grown in DMEM medium (+4.5 g/L D-Glucose, L-glutamine, Thermo Fisher Scientific, Darmstadt, Germany) supplemented with 10% FCS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cells were counted using a Neubauer cell counting chamber (Laboroptik, Friedrichsdorf, Germany). Cells were routinely tested negative for mycoplasma (PCR Mycoplasma Test Kit I/C; PK-CA91-1096; Promocell, Heidelberg, Germany). For coating, the recombinant proteins (acid extracted Bovine Collagen Type I, Curacyte Discovery GmbH, Leipzig, Germany; human fibronectin, Roche Life Science, Penzberg, Germany) were diluted in PBS and incubated overnight at 4 °C.
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6

Isolation and Culture of EPCs

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EPCs were cultured according to the previously described method of Chen et al (2004) (34 ). Mononuclear cells (MNCs) were isolated from peripheral blood of healthy young human volunteers by Ficoll-Paque plus (GE Healthcare) using density gradient centrifugation. MNCs were then plated on culture dishes coated with human fibronectin (Roche) and cultured in VascGrowTM medium (Stem Cell and Cancer Institute) at 37°C in a 5% CO2 incubator. After 4 days of culture, medium were changed and the culture was maintained through day 7. Informed consent was obtained from all volunteers and all procedures performed in this study were approved by Research Ethics Committee of Faculty of Medicine, Maranatha Christian University and Immanuel Hospital, Bandung, Indonesia.
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7

Human Fibronectin-Coated SMC Transfection

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Glass coverslips (Fisher Scientific) were coated with 5 µg/mL human fibronectin (Roche Applied Science) and placed in 24 well plates for two hours. SMC in DMEM 10%BGS 1%P/S were plated at a density of 10 000 cells/well. After 24 hours of incubation, the cells were transfected with 0.8 µg/well of the appropriate DNA in serum free media, following the Lipofectamine 2000 protocol (Invitrogen). After four hours, the media was changed to DMEM 10%BGS 1%P/S. siRNA transfection were done using Dharmafect 2 (Dharmacon) as per manufacturers protocol.
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8

Cell Adhesion and Spreading Assay

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Glass coverslips were coated with 30 µg/ml rat tail collagen I (Corning) or 10 µg/ml human fibronectin (Roche) in PBS overnight at 4 °C. 1 × 104 Cells in serum free medium were plated and left to settle at 37 °C for the indicated time points. Cells were fixed in 4% PFA/PBS. Afterwards, the spread cells were visualized by AlexaFluor-488 conjugated phalloidin (Cell Signaling), DAPI and vinculin staining. Micropatterned glass slides were from Cytoo.
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9

Isolation and Expansion of Endothelial Colony Forming Cells

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Human mononuclear cells (MNCs) were isolated using a procedure modified from Taljaard et al.[29 (link)] MNCs were obtained from a healthy subject using an Optia leukopheresis platform (Key Biologics). The MNC product processed through a density gradient (Ficoll-Paque Premium, GE Healthcare) and rinsed repeatedly with PBS. After counting, cells were plated at 415,000cells cm−2 in endothelial growth media (EGM-2, Lonza) with 20% human serum (Sigma) and onto human fibronectin (Roche) coated-tissue culture flasks (1.6µg fibronectin cm−2). Cells were incubated at 37°C in 5% CO2 and media exchanged every 2–3days. After 14days, media was removed from the flasks, rinsed with PBS, and incubated with 0.08mL cm−2 TrypLE (Invitrogen) to passage the cells. Cells from 3 flasks were combined onto 1 flask without fibronectin coating. After sufficient outgrowth of ECFCs, cells were sorted using magnetic CD31- Dynabeads (Invitrogen), according to the manufacturer’s protocol. The CD31+ population was grown to confluence and frozen in 50% EGM-2, 40% fetal bovine serum (FBS, Hyclone), and 10% dimethyl sulfoxide. After thawing, the ECFCs were maintained in complete endothelial growth medium with 10% FBS and used at P4 or P5 for seeding experiments.
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10

Characterization of Human Endothelial Progenitor Cells

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Human blood samples (n=3) were provided by healthy human volunteers. The EPCs were characterized based on the binding of lectin Ulex europaeus agglutinin-1 (FITC-UEAI) (Sigma-Aldrich, USA) and uptake of acetylated-low density lipoprotein (DiI-Ac-LDL) (Sigma-Aldrich, USA). 2'-7'-dichlorofluorescein diacetate (DCF-DA), 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen, USA), Ficoll-| 40 Paque (GE Healthcare, Sweden), human fibronectin (Roche, Switzerland), VascGrow TM medium (Stem Cell and Cancer Institute, Indonesia), Fc Receptor (FcR) blocker (Miltenyi Biotec, Germany), CD133 PE (Miltenyi Biotec, Germany), VEGFR2/KDR/Flk-1 PE (RandD System, USA). quercetin, kaempferol, myricetin used from (Biopurify Phyto-chemical, China).
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