Human fibronectin

Manufactured by Merck Group

Sourced in United States, United Kingdom, Germany, France, Canada, Macao, Switzerland

Human fibronectin is a high-molecular-weight extracellular matrix glycoprotein that plays a crucial role in cell adhesion, migration, growth, and differentiation. It is a soluble dimeric protein found in a soluble form in the plasma and in an insoluble form in the extracellular matrix.

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Lab products found in correlation

Bovine serum albumin (bsa), by Merck Group (15 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (9 mentions) L glutamine, by Thermo Fisher Scientific (8 mentions) Glutamax, by Thermo Fisher Scientific (8 mentions) Matrigel, by BD (7 mentions) Hepes, by Thermo Fisher Scientific (7 mentions) Poly l lysine (pll), by Merck Group (7 mentions) Fetal bovine serum (fbs), by Merck Group (6 mentions) Transdux reagent, by System Biosciences (6 mentions) Trypsin edta, by Thermo Fisher Scientific (6 mentions) Human collagen 4, by Merck Group (5 mentions) Triton x 100, by Merck Group (5 mentions) Puromycin, by Merck Group (4 mentions) Polycarbonate membrane, by Neuro Probe (4 mentions) Boyden chamber, by Neuro Probe (4 mentions) Poly l ornithine, by Merck Group (4 mentions) Endothelial basal medium 2, by Lonza (4 mentions) Phosphate buffered saline (pbs), by Merck Group (4 mentions) Sodium pyruvate, by Thermo Fisher Scientific (4 mentions)

Close spelling variants of this product

Fibronectin (1886 citations) Human plasma fibronectin (112 citations) Fibronectin from human plasma (24 citations) Human plasma fibronectin (FN) (8 citations) Anti-human fibronectin (4 citations) Fibronectin (FN) from human plasma (4 citations) Human cellular fibronectin (4 citations) Chemicon™ Human Plasma Fibronectin Purified Protein fibronectin solution (4 citations) Rabbit anti-human fibronectin (4 citations) Human plasma fibronectin purified protein (3 citations)

194 protocols using human fibronectin

Microfluidic Endothelial Cell Inflammation Assay

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Prior to cell seeding, the surface of the microfluidic device was coated using 100 μg/ml human fibronectin (Sigma Aldrich, Israel) in PBS. Human umbilical vein endothelial cells (HUVECs), (Lonza, Israel) were used in all assays. HUVECs were cultured in T75/T150 flasks with ECM media supplemented with FBS, Endothelial Cell Growth Supplement, penicillin/streptomycin solution (ScienCell, Switzerland). HUVECs were trypsinized using trypsin B (Biological Industries, Israel), and 10⁶ cells were seeded in each microfluidic channel. The cells were then incubated at 37°C and 5% CO² with standard media for 2 hr to enable a stable cell adhesion.
Inflammation was induced by activating the HUVECs using 0.1 μg/ml TNF‐α (R&D Systems, MN) in a serum free media, which resulted in E‐selectin and ICAM‐1 over‐expression. The activation was induced under static conditions at 37°C and 5% CO₂ over different durations (0.5–6 hr). Then the microfluidic channels were washed twice with PBS. Cells were then fixed using 4% paraformaldehyde (Sigma Aldrich, Israel) for 10 min at room temperature, washed three times and stored at 4°C.

Zukerman H., Khoury M., Shammay Y., Sznitman J., Lotan N, & Korin N. (2019). Targeting functionalized nanoparticles to activated endothelial cells under high wall shear stress. Bioengineering & Translational Medicine, 5(2), e10151.

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Quantification of Focal Adhesions and F-Actin

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Cells were seeded onto human fibronectin (10 µg/ml; Sigma-Aldrich)-coated coverslips. After transfection or incubation overnight, cells were fixed in 4% paraformaldehyde in PBS for 20 min at RT and subsequently permeabilized with 0.2% Triton X-100 in PBS for 5 min. For F-actin staining, cells were incubated with either TRITC- or Alexa Fluor 488–conjugated phalloidin (Invitrogen) and diluted in PBS for 1 h at RT. After this incubation, cells were washed three times in PBS. For detection of paxillin, primary antibodies were diluted in PBS with 3% BSA (Thermo Fisher Scientific) and incubated for 2 h at RT. After labeling with the primary antibodies, cells were washed three times with PBS before incubation with either Alexa Fluor 568– or 488–conjugated secondary antibodies (Invitrogen) and phalloidin. Cells were then imaged using either a microscope (IX71; Olympus) with a 40× oil-immersion objective (UPlanFLN) with a numerical aperture of 1.3 and Image-Pro Plus software (Media Cybernetics) or a confocal laser-scanning microscope (LSM510; Carl Zeiss) with a Plan Fluorite 100× oil objective with a numerical aperture of 1.45 and the accompanying LSM510 software. Focal adhesion number and length were quantified using ImageJ software (National Institutes of Health).

Dart A.E., Box G.M., Court W., Gale M.E., Brown J.P., Pinder S.E., Eccles S.A, & Wells C.M. (2015). PAK4 promotes kinase-independent stabilization of RhoU to modulate cell adhesion. The Journal of Cell Biology, 211(4), 863-879.

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Quantitative Analysis of Extracellular Matrix Proteins

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Primary antibodies were directed against human elastin (rabbit polyclonal antibody, Novotec, France), human fibronectin (rabbit polyclonal antibody, Sigma, France) and human collagen (rabbit polyclonal antibody, Origene, Rockville, MD, USA). Goat anti-rabbit Alexa Fluor 594 (Invitrogen, Waltham, MA, USA) was used as conjugate for elastin. Fluorescent staining of elastin was observed using Cytation 5 cell imaging multi-mode reader and quantified using Gen5 image software (BioTek, Winooski, VT, USA); the results were expressed as RFU (relative fluorescent unit) % (area of fluorescent regions vs. total area)/nuclei number ratio in cells. Goat anti-rabbit Europium and Delfia^® enhancement solutions were used as conjugates (Perkin-Elmer) for fibronectin and collagen. Fluorescent staining of fibronectin and collagen was quantified using a microplate reader (Infinite M1000, TECAN) and results were expressed as RFU (relative fluorescence unit: fluorescent signal) and normalized to the nuclei number.

Rorteau J., Chevalier F.P., Bonnet S., Barthélemy T., Lopez-Gaydon A., Martin L.S., Bechetoille N, & Lamartine J. (2022). Maintenance of Chronological Aging Features in Culture of Normal Human Dermal Fibroblasts from Old Donors. Cells, 11(5), 858.

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Investigating NSCLC Cell Line Responses

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All drugs were purchased from Selleck Chemicals (Houston, TX) and prepared as 10 mmol/l stock solutions in dimethyl sulfoxide. Predesigned sets of four independent siRNA sequences of the target genes Plk1 and MET (siGENOME SMARTpool; Dharmacon, Lafayette, CO) were used. Human TGF‐β1 was purchased from Cell Signaling Technology (Danvers, MA). Human fibronectin was purchased from Sigma‐Aldrich (St. Louis, MO). Constitutive active TPR‐Met plasmid and control pBABE plasmid were obtained from Addgene (Cambridge, MA). Antibodies and dilutions used in the study are listed in Appendix Table S3. The HGF neutralizing monoclonal antibody (24612.111) was acquired from Invitrogen (Catalog # MA1‐24767, Thermo Fisher Scientific, Waltham, MA).
Human NSCLC cell lines were obtained, maintained, and genotyped by STR profiling as previously described (Ferrarotto et al, 2016; Peng et al, 2016) and routinely tested for the presence of mycoplasma species using the Mycoplasma Detection Kit (Lonza, Basel, Switzerland).

Singh R., Peng S., Viswanath P., Sambandam V., Shen L., Rao X., Fang B., Wang J, & Johnson F.M. (2019). Non‐canonical cMet regulation by vimentin mediates Plk1 inhibitor–induced apoptosis. EMBO Molecular Medicine, 11(5), e9960.

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Stable F508del CFTR cell lines for research

Cited 1 time

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A CFBE41o- cell line stably expressing F508del CFTR harboring an HRP-tag in the fourth extracellular loop was obtained from Professor Gergely Lukacs (Department of Physiology, McGill University, Montreal, QC, Canada) (Veit et al., 2012 (link)). Cells were grown in Eagle’s minimal essential medium (MEM) (Life Technologies) supplemented with 10% FBS, 1% L-glutamine (Life Technologies), 10 mM HEPES (Life Technologies), 200 μg/ml geneticin (Life Technologies) and 3 μg/ml puromycin (Sigma) in culture flasks coated with 0.01% bovine serum albumin (BSA) (Sigma), 30 μg/ml Purecol (Nutacon) and 0.001% human fibronectin (Sigma). HEK293 cells were cultured in uncoated flasks using Dulbecco’s Modified Eagle Medium (DMEM) (Life Technologies) supplemented with 10% FBS and 1% penicillin/streptomycin. The U2OS EA-MEM F508delCFTR cell line expresses the larger beta-galactosidase EFC fragment localized in the plasma MEMbrane (EA: enzyme acceptor) and CFTR with the EA-MEM fusion protein, and was obtained from DiscoverX. These cells were cultured in a medium developed by DiscoverX (assay complete medium, 92-0018GK3). CHO cells were cultured in DMEM containing 10% FBS.

de Wilde G., Gees M., Musch S., Verdonck K., Jans M., Wesse A.S., Singh A.K., Hwang T.C., Christophe T., Pizzonero M., Van der Plas S., Desroy N., Cowart M., Stouten P., Nelles L, & Conrath K. (2019). Identification of GLPG/ABBV-2737, a Novel Class of Corrector, Which Exerts Functional Synergy With Other CFTR Modulators. Frontiers in Pharmacology, 10, 514.

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Fibronectin, HGF, and Cytoskeleton Analysis

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Human fibronectin, U0126 and fluorescein isothiocyanate(FITC)-conjugated phalloidin were purchased from Sigma-Aldrich (Gillingham, UK), LY294002 from Merck, recombinant human HGF from R&D Systems (Abingdon, UK), antibodies against pAKT [Ser473], AKT and pERK from Cell Signaling Technology, ERK1 (K-23), Met (C-12, SP260), protein A/G PLUS-agarose were from Santa Cruz Biotechnology (Santa Cruz, CA), and Immobilon P membrane from Millipore.

Pajari A.M., Päivärinta E., Paavolainen L., Vaara E., Koivumäki T., Garg R., Heiman-Lindh A., Mutanen M., Marjomäki V, & Ridley A.J. (2016). Ellagitannin-rich cloudberry inhibits hepatocyte growth factor induced cell migration and phosphatidylinositol 3-kinase/AKT activation in colon carcinoma cells and tumors in Min mice. Oncotarget, 7(28), 43907-43923.

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Establishing Cell-Derived ECM Co-Cultures

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NuFF (passages 18–28) were co-cultured with MDA breast cancer cells for establishment of cell-derived ECM. Both cell lines were seeded on the same day in a 1:1 ratio in 4 well Nunc LabTek II Chamberslides (Sigma) using methods previously described [11 (link), 14 (link)]. In brief, co-cultures were maintained in one-half NuFF media and one-half MDA media with a final concentration of 10% fetal bovine serum (FBS). In some instances, chamberslides were coated with 5ug/well human fibronectin (Sigma) for 1 hour at 37°C, 5% CO₂ in a humidified atmosphere prior to cell seeding. Media was exchanged every 2–3 days and co-cultures were maintained for 7–9 days before de-cellularization.

Hielscher A., Ellis K., Qiu C., Porterfield J, & Gerecht S. (2016). Fibronectin Deposition Participates in Extracellular Matrix Assembly and Vascular Morphogenesis. PLoS ONE, 11(1), e0147600.

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Live-Cell Imaging of Neutrophil Chemotaxis

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For live-cell imaging, HL-60 neutrophils were collected, washed and resuspended with mHBSS containing 0.2% BSA, and then seeded on Lab-Tek chambered cover glasses (NUNC) coated with 50 μg/ml human fibronectin (Sigma-aldrich). Time-lapse microscopy was performed on a Leica Spinning Disk Confocal microscope. Chemoattractant fMLP (Sigma) was given to cells either globally or delivered by a Femtotips micropipette (Eppendorf). Total Internal Reflection Fluorescence (TIRF) microscopy was performed using a Nikon Eclipse Ti-E TIRF microscope. Fluorescence recovery after photobleaching (FRAP) was performed on Zeiss Axiovert 200 Laser scanning microscope with LSM510-Meta confocal module. For immuno uorescent staining, cells were xed, and then stained with Anti-PI 3-Kinase, p110γ (clone 17D7.2, Merck Millipore) in PBS containing 1% BSA as indicated in the Extended Experimental Procedures. Images were analyzed with ImageJ (NIH) as described in the Extended Experimental Procedures.

Wang M., Artemenko Y., Cai W., Iglesias P.A, & Devreotes P.N. (2014). The Directional Response of Chemotactic Cells Depends on a Balance between Cytoskeletal Architecture and the External Gradient. Cell reports, 9(3), 1110-1121.

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Padlock Assay for HeLa Kyoto Cells

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HeLa Kyoto cells were cultured according to standard protocols in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Padlock assays were carried out in 384-well microplates with glass bottoms (Corning). Prior to cell seeding, wells were incubated with 25 µg/mL human fibronectin (Sigma) diluted in PBS for 30 min. Cells were then trypsinized and resuspended in DMEM at a concentration of 0.15–0.2 × 10⁶ cells/mL, and 10 µL of cell suspension was applied to each well. After a 10 min pre-incubation, the total volume of DMEM was increased to 60 µL per well and cells grown overnight at 37°C and 5% CO₂.

Schneider N, & Meier M. (2017). Efficient in situ detection of mRNAs using the Chlorella virus DNA ligase for padlock probe ligation. RNA, 23(2), 250-256.

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Isolation and Culture of Human Adipose-Derived Mesenchymal Stem Cells

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After tumescent liposuction, lipoaspirates were centrifuged at 3×10³g for 3 minutes and the stromal vascular fraction was collected in a sterile container. The samples were enzymatically dissociated in a 0.05% Collagenase type II solution (Sigma-Aldrich) in Joklik modified Eagle’s Medium (Sigma-Aldrich) for 20 minutes at 37°C. Collagenase activity was stopped by the addition of 0.1% BSA (Sigma-Aldrich) solution in Joklik modified Eagle’s Medium (Sigma-Aldrich). Cell suspension was centrifuged at 1×10³g for 10 minutes. Samples collected from different subjects were kept distinct and used to obtain distinct hAT-MASC cell lines.
2.0×10⁶ freshly isolated human cells were plated onto 100 mm human fibronectin (Sigma-Aldrich) coated dishes (BD Falcon) in an expansion medium composed as follows: 60% low glucose DMEM (Invitrogen), 40% MCDB-201, 1 mg/mL linoleic acid-BSA, 10⁻⁹M dexamethasone, 10⁻⁴M ascorbic acid-2 phosphate, 1X insulin-transferrin-sodium selenite (all from Sigma-Aldrich), 2% fetal bovine serum (StemCell Technologies), 10 ng/ml human or murine PDGF-BB, 10 ng/ml human or murine EGF (both from Peprotech EC). Medium was replaced with fresh one every 4 days. Once cells reached 70–80% of confluence, they were detached with 0.25% trypsin-EDTA (Sigma-Aldrich) and re-plated at a density of 1–2×10³/cm².

Domenis R., Bergamin N., Gianfranceschi G., Vascotto C., Romanello M., Rigo S., Vagnarelli G., Faggiani M., Parodi P., Kelley M.R., Beltrami C.A., Cesselli D., Tell G, & Beltrami A.P. (2014). The Redox Function of APE1 Is Involved in the Differentiation Process of Stem Cells toward a Neuronal Cell Fate. PLoS ONE, 9(2), e89232.

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