Human fibronectin

Manufactured by R&D Systems

Sourced in Taiwan, Province of China

Human fibronectin is a high molecular weight glycoprotein found in the extracellular matrix and in plasma. It plays a role in cell adhesion, cell motility, and wound healing.

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Fibronectin (69 citations) Recombinant human fibronectin (5 citations) Human fibronectin protein (2 citations) Human Fibronectin DuoSet ELISA (2 citations) Human fibronectin ELISA kit (2 citations) Human plasma fibronectin (2 citations)

9 protocols using human fibronectin

Measuring Fibroblast Response to TGF-β1

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Sub‐confluent lung fibroblasts grown in 6‐well plates were deprived of serum for 2 h and stimulated with or without 10 pm TGF‐β1. The supernatant from the cultured cells was harvested after 24 h and stored at −80 °C until analysis. Fibronectin, TGF‐β1, and COL11A1 production by the cells were determined using human Fibronectin (R&D Systems), TGF‐β1 (R&D Systems), and COL11A1 (Abnova, Taipei, Taiwan) ELISA kits, respectively, according to the manufacturers' instructions.

Iwai M., Tulafu M., Togo S., Kawaji H., Kadoya K., Namba Y., Jin J., Watanabe J., Okabe T., Hidayat M., Sumiyoshi I., Itoh M., Koyama Y., Ito Y., Orimo A., Takamochi K., Oh S., Suzuki K., Hayashizaki Y., Yoshida K, & Takahashi K. (2021). Cancer‐associated fibroblast migration in non‐small cell lung cancers is modulated by increased integrin α11 expression. Molecular Oncology, 15(5), 1507-1527.

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Transwell Boyden Chamber Migration Assay

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The migrations of MCF-7 and MDA-MB-231 cells transfected with different miRNAs or plasmids were tested in a Transwell Boyden Chamber (6.5 mm, Costar, Cambridge, MA). The polycarbonate membranes (8-μm pore size) on the bottom of the upper compartment of the transwells were coated with 1% human fibronectin (R&D systems). Cells were harvested 48 hours later after transfection, and suspended in FBS-free DMEM or L-15 culture medium. Then cells were added to the upper chamber (4 × 104 cells/well). At the same time, 0.5 ml of DMEM or L-15 with 10% FBS was added to the lower compartment, and the plates were incubated for 8–12 hours in a 5% CO₂ atmosphere saturated with H₂O. After incubation, cells that had entered the lower surface of the filter membrane were fixed with 4% paraformaldehyde for 25 minutes at room temperature, washed 3 times with distilled water, and stained with 0.1% crystal violet in 0.1 M borate and 2% ethanol for 15 minutes at room temperature. Cells remaining on the upper surface of the membranes were scraped off gently with a cotton swap. The upper surfaces with migrant cells were captured 5–6 fields per chamber by a photomicroscope (BX51, Olympus, Japan), and the number of cells were counted blindly.

Pan Y., Li J., Zhang Y., Wang N., Liang H., Liu Y., Zhang C.Y., Zen K, & Gu H. (2016). Slug-upregulated miR-221 promotes breast cancer progression through suppressing E-cadherin expression. Scientific Reports, 6, 25798.

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FXIIIa-Mediated Crosslinking of ECM Proteins

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We hypothesized that large-sized ECM proteins could be natural substrates for FXIIIa and would tether to the hydrogel network without further conjugation. ECM proteins of interest were purchased in solution form: mouse laminin 1, collagen IV (both mouse, BD Biosciences) and human fibronectin (human, R&D Systems). We performed a fluorescent binding assay, in which proteins were mixed with each of the fluorescent FXIIIa-substrates (Q-peptide-Alexa647 or Lys-Tamra) in the presence of FXIIIa. The reactions were qualitatively analysed by SDS–PAGE and in-gel fluorescence scanning, demonstrating that indeed the proteins are susceptible for FXIIIa-based cross-linking (Supplementary Fig. 1C). All ECM proteins were aliquoted at 4C and stored at −20 °C.

Ranga A., Gobaa S., Okawa Y., Mosiewicz K., Negro A, & Lutolf M.P. (2014). 3D niche microarrays for systems-level analyses of cell fate. Nature Communications, 5, 4324.

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Monocyte Adhesion Assay on Coated Substrates

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Adhesion assays were performed either in plastic 96-well plates (flat bottom) or on glass coverslips. The coating of the substrate was done with 150 μg/mL of collagen, type I solution from rat tail (Sigma), 12.5 μg/mL of recombinant Human ICAM-1/CD54 (R&D Systems), or 5 µg/cm² of human fibronectin from R&D Systems. Primary monocytes were added to the coated substrate and incubated for 2 h at 37 °C. Afterward, the cells were processed for immunofluorescence staining with Phalloidin A568 (Invitrogen), or analyzed for cell viability with the CellTiter-Glo Luminescent Cell Viability Assay (Promega).

Ayala-Nunez N.V., Follain G., Delalande F., Hirschler A., Partiot E., Hale G.L., Bollweg B.C., Roels J., Chazal M., Bakoa F., Carocci M., Bourdoulous S., Faklaris O., Zaki S.R., Eckly A., Uring-Lambert B., Doussau F., Cianferani S., Carapito C., Jacobs F.M., Jouvenet N., Goetz J.G, & Gaudin R. (2019). Zika virus enhances monocyte adhesion and transmigration favoring viral dissemination to neural cells. Nature Communications, 10, 4430.

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Molecular Signaling Pathway Analysis

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BAY 60-2770 and BAY 41-2272 were provided by Bayer AG (Wuppertal, Germany). 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was purchased from Cayman Chemical (Ann Arbor, MI), and hemin was obtained from Frontier Scientific (Newark, NJ). Recombinant human and murine tumor necrosis factor-α (TNF) and human fibronectin were from R&D Systems (Minneapolis, MN). Dulbecco’s modified Eagle’s medium, fetal bovine serum, penicillin, and streptomycin were from Gibco-Invitrogen (New York, NY). Trizol was from Invitrogen (Carlsbad, CA). All other reagents were from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

Ferreira WA J.r., Chweih H., Lanaro C., Almeida C.B., Brito P.L., Gotardo E.M., Torres L., Miguel L.I., Franco-Penteado C.F., Leonardo F.C., Garcia F., Saad S.T., Frenette P.S., Brockschnieder D., Costa F.F., Stasch J.P., Sandner P, & Conran N. (2020). Beneficial Effects of Soluble Guanylyl Cyclase Stimulation and Activation in Sickle Cell Disease Are Amplified by Hydroxyurea: In Vitro and In Vivo Studies. The Journal of Pharmacology and Experimental Therapeutics, 374(3), 469-478.

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Characterization of hiPSC-Derived Cardiomyocytes

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hiPSC-CM (NCardia) were cultured following the manufacturer’s protocol and
proprietary media at a cell density of 100 000 cells cm⁻². Prior to
plating, multiwell groove array was coated with human fibronectin (10
μg ml⁻¹, R&D Systems) for 1 h, then washed
twice with phosphate buffered saline (PBS, Sigma-Aldrich). On day 10, cells were
loaded with the voltage sensitive fluorescent dye FluoVolt (1:1000,
ThermoFisher) along with Powerload (1:100, ThermoFisher) in serum-free
medium.and incubated for 25 min at 37 °C. Subsequently, action potentials were
recorded using the CellOPTIQ^® system (Clyde Biosciences) at 10 000
fps as the depolarization time of cardiomyocytes is between 5 and 10 ms.
Additionally, contractility analysis was done by recording videos at 100 fps
that were analyzed using the MuscleMotion software [69 (link)].

Huethorst E., Cutiongco M.F., Campbell F.A., Saeed A., Love R., Reynolds P.M., Dalby M.J, & Gadegaard N. (2020). Customizable, engineered substrates for rapid screening of cellular cues. Biofabrication, 12(2), 025009.

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Cell Adhesion Assay with Peptides

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The assays were performed following a protocol described elsewhere (17 (link)). Cells resuspended in DMEM containing 1% BSA were treated with peptides for 30 minutes at 37°C under mild rotation. The cells were seeded in 96-well plates coated with human fibronectin (R&D Systems) or bovine collagen type-I (BioPioneer, San Diego, CA) at 4 × 10⁵ cells/ml (LM-PmC) or 2 × 10⁵ cells/ml (GFP-PC-3), and allowed to attach to the wells for 30 minutes at 37°C in a CO₂ incubator in the presence of the peptides. In some cases, the cells were treated with the peptides in the presence of 10 μg/ml of anti-NRP-1 b1b2 or control IgG, or treated solely with anti-human β1 (EMD Millipore) or anti-mouse β1 (eBioscience) integrin subunit antibodies or control IgG (Abcam). After the incubation, the cells were treated with 0.25% crystalline trypsin for 2 minutes, and the reaction was stopped by adding 0.5 mg/ml soybean inhibitor. The wells were gently washed with warm DMEM to remove detached cells, and the cells that remained attached to the wells were cultured in full DMEM containing 0.5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Two hours later, the cells were lysed with a buffer containing dimethylsulfoxide and methanol at 1:1 volume ratio, and absorbance at 595 nm was read with a microplate reader.

Sugahara K.N., Braun G.B., de Mendoza T.H., Kotamraju V.R., French R.P., Lowy A.M., Teesalu T, & Ruoslahti E. (2014). Tumor-Penetrating iRGD Peptide Inhibits Metastasis. Molecular cancer therapeutics, 14(1), 120-128.

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Adhesion Assay of DOX-Treated Cells

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MGC‐803‐CD2AP cells were pretreated with 1 µg/mL of DOX for 48 hours, and incubated in a 24‐well plate precoated with 2 μg/ml human fibronectin (R&D Systems, Minneapolis, MN) for 2 hours at 37°C. The fibronectin was removed, and the cells were washed with PBS three times. The pretreated cells were then seeded in 24‐well plates (1 × 10³ cells/well) and incubated for 1 hour at 37°C. The nonadherent cells were washed off with PBS three times, and the remaining adherent cells were fixed with ice‐cold methanol, stained with crystal violet, and counted under a microscope. Three representative fields were randomly counted for analysis.

Xie W., Chen C., Han Z., Huang J., Liu X., Chen H., Zhang T., Chen S., Chen C., Lu M., Shen X, & Xue X. (2020). CD2AP inhibits metastasis in gastric cancer by promoting cellular adhesion and cytoskeleton assembly. Molecular Carcinogenesis, 59(4), 339-352.

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Extracellular Matrix Protein Preparation

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Human fibronectin and vitronectin were purchased from R&D Systems (Minneapolis, MN), human laminin from Millipore (Temecula, CA), β-1,6-glucanase from Takara Bio Inc. (Otsu, Shiga, Japan), β-1,3-glucanase (lyticase) from Sigma and α1-2,3 mannosidase and α1-6 mannosidase from New England Biolabs (Ipswich, MA). Proteinase K and bovine serum albumin (BSA) were obtained from BioShop Canada Inc. (Burlington, Ontario, Canada), trypsin from Promega (Madison, WI) or Biocentrum (Krakow, Poland) and horseradish peroxidase-conjugated streptavidin solution (SA-HRP) from MP Biomedicals (Solon, OH).

Kozik A., Karkowska-Kuleta J., Zajac D., Bochenska O., Kedracka-Krok S., Jankowska U, & Rapala-Kozik M. (2015). Fibronectin-, vitronectin- and laminin-binding proteins at the cell walls of Candida parapsilosis and Candida tropicalis pathogenic yeasts. BMC Microbiology, 15, 197.

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