Human fibronectin

Manufactured by Thermo Fisher Scientific

Sourced in United States, Canada

Human fibronectin is a high-molecular-weight glycoprotein found in the extracellular matrix and body fluids. It plays a critical role in cell adhesion, migration, growth, and differentiation.

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Fibronectin (342 citations) Human plasma fibronectin (30 citations) Fibronectin from human plasma (4 citations) Human fibronectin protein (2 citations)

46 protocols using human fibronectin

Visualizing Cytoskeletal Structures in Cells

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Cells were resuspended in culture media containing 0.5 mM MnCl₂ and seeded on a coverslip glass-bottom tissue culture dish (MatTek, Ashland, MA) that was precoated with human fibronectin (Gibco) at concentration of 10 μg/mL. Cells were allowed to adhere on fibronectin under culture conditions for 2 hours. Medium was discarded, and cells were fixed in 3.7% (w/v) paraformaldehyde-PBS solution at RT for 10 minutes. Cells were then incubated in CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 1 mM EGTA, 10 mM PIPES [pH 6.8]) containing 0.3% (v/v) Triton at RT for 1 minute. Cells were washed 3 times with PBS and nonspecific sites blocked with PBS containing 5% (w/v) BSA overnight at 4 °C. Fixed cells were stained with DAPI and Alexa Fluor 488 phalloidin (Invitrogen Corporation, Carlsbad, CA). Cells were visualized under confocal laser scanning microscope LSM710 (Zeiss, Oberkochen, Germany) at a magnification of 40× and 100× under oil immersion.

Bu W., Levitskaya Z., Loh Z.Y., Jin S., Basu S., Ero R., Yan X., Wang M., Ngan S.F., Sze S.K., Tan S.M, & Gao Y.G. (2020). Structural basis of human full-length kindlin-3 homotrimer in an auto-inhibited state. PLoS Biology, 18(7), e3000755.

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Cell Adhesion Assay Protocols

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Cell adhesion assays were performed using 96-well round-bottomed microtiter plates (Immulon-2HB; Dynex Technologies, Inc.). The wells were coated overnight at 4°C with 10 μg/ml human fibronectin (GIBCO BRL), collagen I (Nitta Gelatin Co., Japan), laminin 111 (laminin 1; Invitrogen), laminin 211 (laminin 2; Invitrogen), and human laminin 511/521 (laminin10/11; R&D Systems), and then diluted with PBS and blocked with 3% bovine serum albumin for 1 hour at 37°C. After washing, 10,000 cells were added and incubated for 60 minutes at 37°C. Antibodies against β1, α6, and α4 integrins, or the RGD and RAD peptides were added to the culture medium as potential inhibitors of cell adhesion. The plates were washed 3 times with PBS to remove unattached cells, and then the attached cells were identified by staining with crystal violet and evaluated using spectrophotometric analysis (wavelength; 600 nm).

Saito K., Fukumoto E., Yamada A., Yuasa K., Yoshizaki K., Iwamoto T., Saito M., Nakamura T, & Fukumoto S. (2015). Interaction between Fibronectin and β1 Integrin Is Essential for Tooth Development. PLoS ONE, 10(4), e0121667.

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Endothelial Activation Assay with Fluorescence Imaging

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Endothelial activation effects of the materials were determined
using fluorescence imaging. The HPMECs were seeded onto 12 well plates
containing glass coverslips coated with human fibronectin (25 ug/ml) (Gibco)
with a seeding density of 1.5×10⁴ cells/cm².
The cells were fed with CM on days one, three and six in a similar manner to
that used for the cytotoxicity experiments. Cells were also grown with
regular cell media in wells. For a positive control, 50 ng/ml of
IL1-β (PeproTech) was added to the cells grown with regular growth
media on day six to activate the HPMEC. On day seven, glass slides were
fixed and permeabilized with 0.5% Triton X-100 in 3% methanol-free
paraformaldehyde. For immunofluorescence labeling, rabbit polyclonal
anti-VE-cadherin (1:100) (Cayman Chemicals) and mouse monoclonal
anti-phosphotyrosine (1:50) (BD) were used as primary antibodies. Alexa
Fluor 488 conjugated donkey anti-rabbit IgG (1:100)(Life Technologies) and
cy3 goat anti-mouse IgG (1:100) (Jackson ImmunoResearch) were used as
secondary antibodies. Conjugated F-actin 647 (1:50) (Life Technologies) and
DAPI (1:50) (4′,6-diamidino-2-phenylindole; Thermo Fisher Scientific)
were also used. Slides were imaged with a Nikon Eclipse TE200 microscope
using Volocity software (Quorum Technologies).

Bachtiar E.O., Erol O., Millrod M., Tao R., Gracias D.H., Romer L.H, & Kang S.H. (2020). 3D printing and characterizations of a soft and biostable elastomer with high flexibility and strength for biomedical applications. Journal of the mechanical behavior of biomedical materials, 104, 103649.

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Topical Nanoparticle Formulation Development

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Quercetin, epigallocatechin gallate (EGCG), poloxamer 407, and hyaluronic acid sodium salt (HA) with Mws of 8000–15,000 were purchased from Myskinrecipe (Bangkok, Thailand). Poly(DL-lactic-co-glycolic acid) (50:50) with a terminal carboxyl group (PLGA, inherent viscosity 0.22 dL/g, molecular weight of 6700 Da) was purchased from Lactel Absorbable Polymers (Birmingham, AL, USA). MTT was purchased from Roche Applied Science (Penzberg, Germany). Keratinocyte serum-free medium (SFM), insulin, human recombinant zinc solution, collagen I bovine, human fibronectin, Dulbecco’s phosphate-buffered saline (PBS), fetal bovine serum (FBS), penicillin-streptomycin, trypan blue stain 0.4%, 0.25% trypsin-EDTA, and fluorobrite DMEM were purchased from Gibco (Waltham, MA, USA). The HCE cell line, supplements for keratinocyte consisting of epidermal growth factor (EGF), and bovine pituitary extract (BPE) were purchased from ATCC (Manassas, VA, USA)). Hydrocortisone solution and DPPH (2,2-diphenyl-1-picrylhydrazyl) were purchased from Sigma-Aldrich (St. Louis, MO, USA)). ROS/superoxide reagent kit was purchased from Abcam (Waltham, MA, USA).

Chittasupho C., Junmahasathien T., Chalermmongkol J., Wongjirasakul R., Leesawat P, & Okonogi S. (2021). Suppression of Intracellular Reactive Oxygen Species in Human Corneal Epithelial Cells via the Combination of Quercetin Nanoparticles and Epigallocatechin Gallate and In Situ Thermosensitive Gel Formulation for Ocular Drug Delivery. Pharmaceuticals, 14(7), 679.

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Neonatal Rat Cardiac Cell Culture

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Hearts isolated from neonatal 2-day-old Wistar rats were minced and enzymatically digested using collagenase (Type-1, Wako). The isolated cells were collected by centrifugation and preplated for 1 h. The cells were plated on 22-mm-diameter glass coverslips coated with human fibronectin (12 g/ml, Gibco) at a cardiac cell density of 2.63 × 10³ cells/mm². Cell constructs were incubated in Dulbecco’s Modified Eagle’s Medium (Gibco) with 10% fetal bovine serum (Gibco) and 1% penicillin streptomycin (Sigma) for 24 h under humidified conditions at 37 °C and 5% CO₂. The medium was then replaced with a contraction medium, Minimum Essential Medium (Gibco) with 10% calf serum (Gibco), 1% penicillin streptomycin, and 1% cytosine arabino-furanoside (ARA-C, Sigma). The latter prevents overproliferation of contaminating nonmyocytes^{37 (link)} and thus stabilizes cardiac conduction^{38 (link)}.

Hörning M., Blanchard F., Isomura A, & Yoshikawa K. (2017). Dynamics of spatiotemporal line defects and chaos control in complex excitable systems. Scientific Reports, 7, 7757.

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Cell Culture Protocols for Liver Cancer and Healthy Cells

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Hep3B and HuH-7 HCC cell lines (a gift from Professor Peter J. Leedman’s Laboratory) were cultured in DMEM low glucose-pyruvate supplemented containing 10% HyClone fetal bovine serum (FBS) (GE Healthcare Life Sciences, Cat. No. SH30084.03) and 1% antibiotic–antimycotic (Gibco, Cat. No. 15240062). HEK293T cells (ATCC, Cat. No. CRL-3216) were grown in DMEM high glucose-pyruvate containing 10% FBS and 1% antibiotic–antimycotic. The immortalized human liver cell line THLE-3 (ATCC, Cat. No. CRL-1123) was grown in BEBM supplemented with BEGM Bullet Kit (Lonza, Cat. No. CC-3170), from which we omitted the gentamycin/amphotericin and epinephrine, and to which we added 5 ng/mL human epidermal growth factor (EGF) recombinant protein (Gibco, Cat. No. PHG0311), 70 ng/mL O-Phosphorylethanolamine (Sigma-Aldrich, Cat. No. P0503), and 10% FBS. These cells were maintained in flasks pre-coated with a mixture of 0.01 mg/mL human fibronectin (Gibco, Cat. No. 33016015), 0.03 mg/mL bovine collagen type I (Australian Biosearch, Cat. No. 5005-B), and 0.01 mg/mL bovine serum albumin (Sigma-Aldrich, Cat. No. A9647) dissolved in BEBM medium (Lonza). All cell lines used in this study were Mycoplasma-free and maintained at 37 °C and 5% CO₂.

Sgro A., Cursons J., Waryah C., Woodward E.A., Foroutan M., Lyu R., Yeoh G.C., Leedman P.J, & Blancafort P. (2023). Epigenetic reactivation of tumor suppressor genes with CRISPRa technologies as precision therapy for hepatocellular carcinoma. Clinical Epigenetics, 15, 73.

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Myoblasts Differentiation on Fibronectin

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Primary Human Skeletal Muscle Myoblasts (Lonza CC-2580) were seeded at a density of 0.24 × 10⁶ cells/cm² onto Human Fibronectin (Gibco 33016015) coated Transwells^® (Corning 3470) in SkGM™-2 Skeletal Muscle Cell Growth Medium-2 (Lonza CC-3245). Transwell^® inserts contained 150μL apical volume and 1 mL basal volume After 24 hours, myoblasts were changed to differentiation media (DMEM-F12 with 2% Horse serum) for an 4 days, changing media every 48 hours, and subsequently returned to SkGM™-2 Skeletal Muscle Cell Growth Medium-2 at the start of the experiment.

Edington C.D., Chen W.L., Geishecker E., Kassis T., Soenksen L.R., Bhushan B.M., Freake D., Kirschner J., Maass C., Tsamandouras N., Valdez J., Cook C.D., Parent T., Snyder S., Yu J., Suter E., Shockley M., Velazquez J., Velazquez J.J., Stockdale L., Papps J.P., Lee I., Vann N., Gamboa M., LaBarge M.E., Zhong Z., Wang X., Boyer L.A., Lauffenburger D.A., Carrier R.L., Communal C., Tannenbaum S.R., Stokes C.L., Hughes D.J., Rohatgi G., Trumper D.L., Cirit M, & Griffith L.G. (2018). Interconnected Microphysiological Systems for Quantitative Biology and Pharmacology Studies. Scientific Reports, 8, 4530.

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Isolation and Culture of Mouse-derived ECs and BMSCs

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The femur and tibia of 12-week-old male C57BL/6 mice (from Nanjing Medical University Experimental Animal Center, Jiangsu, China) were separated. Washed out by complete culture medium: 10% FBS (ScienCell), 1% penicillin/streptomycin (Hyclone) in LG-DMEM (Gibco), bone marrow was transferred to a 100 mm diameter petri dish and was cultured at 37 °C, 5% CO₂ for 2 days. The suspended cells were collected, centrifuged at 1000 rpm for 5 min, and further cultured in EGM-2MV medium (Lonza) on the petri dish pre-coated with 10 μg /mL human fibronectin (Gibco) to get ECs. And the adherent cells (BMSCs) were cultured with complete culture medium [18 (link)]. The medium was changed every two days and digestion was performed when the confluence was 80% for passaging.

Gu L., Wang Z., Gu H., Wang H., Liu L, & Zhang W.B. (2023). Atf4 regulates angiogenic differences between alveolar bone and long bone macrophages by regulating M1 polarization, based on single-cell RNA sequencing, RNA-seq and ATAC-seq analysis. Journal of Translational Medicine, 21, 193.

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Neutrophil Adhesion on Tunable Substrates

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Briefly, polyacrylamide gel substrates were polymerized on a 25 millimeters (mm) glass coverslip, using 3% acrylamide and 0.2% bisacrylamide for a Young’s modulus of E = 1.5 kPa, along with fluorescent red 0.5

μ m

carboxylate-modified polystyrene beads. Gel substrates were then coated with human fibronectin (Gibco 33016015) using the photoactivatable crosslinker sulfo-SANPAH (Sigma 803332) and rinsed extensively. Further experimental details are documented elsewhere in Oakes et al.^{73 (link)} and Witt et al.^{60 (link)}, respectively. The polyacrylamide gel and coverslip were mounted in a coverslip holder, then covered with 1 mL of Leibovitz L-15 media. About 50,000 neutrophils were plated and allowed to adhere for 15 minutes. Approximately 20-60 adherent cells were imaged in phase microscopy using a Nikon TI-2 epifluorescent microscope using a 40X air objective with a 0.6 numerical aperture. An Okolab enclosure around the TI-2 maintained the apparatus at 37° and 5% CO₂ for the duration of the experiments. Only adherent cells were selected for imaging. The

N

represents the number of individual neutrophils imaged and analyzed, with an

n > 3

for individual septic or healthy donors.

Winn-Nuñez E.T., Witt H., Bhaskar D., Huang R.Y., Reichner J.S., Wong I.Y, & Crawford L. (2024). Generative modeling of biological shapes and images using a probabilistic -shape sampler. bioRxiv.

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Characterization of Leukocyte Adhesion

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Mouse anti-FLAG M2 antibody (Sigma), HRP-conjugated anti-FLAG antibody (Sigma), APC PSGL-1 antibody (FLEG) (ThermoFisher), Human Fibronectin (ThermoFisher), DMSO (Sigma), Phalloidin Alex647 (ThermoFisher), RIPA buffer (Sigma), Phorbol 12-Myristate 13-Acetate (PMA, sigma), SLeX (Sigma), Biotinylated ECL (Vector Laboratories), PNGase F (New England Biolabs), Dihydrorhodamine 123 (DHR123) (ThermoFisher), ProLong Gold Antifade Mountant (ThermoFisher), HRP-conjugated streptavidin (ThermoFisher).

Chen C., Yang C, & Barbieri J.T. (2019). Staphylococcal Superantigen-like protein 11 mediates neutrophil adhesion and motility arrest, a unique bacterial toxin action. Scientific Reports, 9, 4211.

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