Fibronectin

Manufactured by Corning

Sourced in United States, Germany

Fibronectin is a cell-adhesive glycoprotein that plays a crucial role in cell attachment, migration, and differentiation. It is a key component of the extracellular matrix and is commonly used in cell culture applications to promote cell adhesion and growth.

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Human fibronectin (50 citations) Fibronectin-coated 24-well plates (4 citations) Fibronectin-coated filter (4 citations) Fibronectin-coated plates (4 citations) Fibronectin (FN, (3 citations) Fibronectin-coated transwell filters (3 citations) Fibronectin solution (3 citations) Fibronectin-coated wells (2 citations) Bovine fibronectin (2 citations) Fibronectin-coated dishes (2 citations)

172 protocols using fibronectin

Fibronectin-Mediated Cell Adhesion Assay

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Briefly, a fibronectin (Sigma-Aldrich Corporation) solution was previously prepared and stored at 4 ° C. Then, 96-well plates (Costar-3599, Corning, US) were coated with fibronectin (10 μg/ml in PBS) at room temperature for 1 h. After coating, the fibronectin solution was removed. Thermally denatured BSA was added to the plates, followed by an incubation of 1 h at 37 ° C. Then, the plates were washed twice with serum-free RPMI 1,640 medium. CXCL12 (0, 10, 100, and 1,000 ng/ml) was added to the wells of fibronectin-coated plates. After the addition of the prepared 100 µl cell suspension (5 ×10⁵ cells per ml), the plates were incubated at 37 ° C for 30 min and then washed thrice with PBS to remove non-adherent cells. Next, a 10 µl Cell Counting Kit-8 (CCK-8) solution (Dojindo, Japan) was added to each well and incubated for 1.5 h. Finally, the adherent cells were stained and quantified at OD450 using a Microplate Reader (Thermo) according to the manufacturer’s instructions. The cell adhesion ratio of the assay was calculated according to the following formula: cell adhesion (%) = OD of the adhered cells /OD of the total cells ×100%.

Wang Y., Li H, & Li F. (2020). ELMO2 association with Gαi2 regulates pancreatic cancer cell chemotaxis and metastasis. PeerJ, 8, e8910.

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Inhibition of MMP-12 by Fab Fragments

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Fluorescein isothiocyanate (FITC)-conjugated elastin (AnaSpec; 10 μg/ml) was incubated with 7 nM cdMMP-12 D4/wt and 2–2,000 nM purified Fab in 50 mM Tris-HCl, 150 mM NaCl, 5 mM CaCl₂, 100 μM ZnCl₂ pH 7.5. Generated fluorescent signals were measured at 12 hr with λ_Ex of 490 nm and λ_Em of 520 nm. For inhibition assays on fibronectin, 200 nM fibronectin (Corning) was incubated with 200–350 nM cdMMP-12 wt and 2 μM GM6001 or 0.1–6 μM Fabs in 50 mM HEPES, 150 mM NaCl pH 7.5 at 37°C for 2–4 hr. The samples were separated on 5% nonreducing SDS-PAGE and analyzed by western blot analysis with anti-fibronectin antibody (Abcam) and chemiluminescent substrate (Thermo Fisher Scientific). For inhibition assays on modified TEM-1 carrying cleavable sequence (PLGLEEAK) and an N-terminal HA tag, 250-nM modified TEM-1 was incubated with 700 nM cdMMP-12 wt and 10 μM Fabs in 50 mM HEPES, 150 mM NaCl pH 7.5 at 37°C for 4 hr. The samples were separated on 12% nonreducing SDS-PAGE, and the modified TEM-1 was visualized by western blot analysis using anti-HA antibody (Invitrogen) and chemiluminescent substrate. Assays without cdMMP-12s or Fabs were performed as controls.

Lee K.B., Dunn Z.S., Lopez T., Mustafa Z, & Ge X. (2020). Generation of highly selective monoclonal antibodies inhibiting a recalcitrant protease using decoy designs. Biotechnology and bioengineering, 117(12), 3664-3676.

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Hydrogel and Silicone Gel Substrates for Microglia Culture

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The hydrogel was prepared using the Hystem Cell Culture Scaffold Kit (Sigma) according to the manufacturer’s protocol. Briefly, the Hystem stock solution was mixed with Extralink stock solution (crosslinker) along with 10 µg/ml concentration of fibronectin (Corning). The mixture was then poured into a 12-well plate and incubated at 37 °C for gelation to occur. The rigidity of the hydrogel was modified by changing the concentration of the crosslinker to obtain approximate gel stiffnesses of 60 and 600 Pa. The gels were pre-incubated with media and microglia were plated on top of the gel and allowed to spread overnight.
Silicone gels in 6-well plates were commercially purchased from Softsubstrates.com. The gels were coated with 10 µg/ml of fibronectin (Corning). The gels were pre-incubated with media and microglia were plated on top of the gel and allowed to spread overnight.

Dudiki T., Meller J., Mahajan G., Liu H., Zhevlakova I., Stefl S., Witherow C., Podrez E., Kothapalli C.R, & Byzova T.V. (2020). Microglia control vascular architecture via a TGFβ1 dependent paracrine mechanism linked to tissue mechanics. Nature Communications, 11, 986.

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Monitoring Live Toxoplasma Egress Dynamics

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Glass bottom 60 mm dishes (MatTek, catalog# P35G-1.5-14-C) were coated with 50 µg/mL fibronectin (Corning, catalog# 354008) as per the manufacturer’s protocol and then HCT-8 cells were grown to > 95% confluence before infecting cells in each dish with 48 × 10⁴ oocysts triggered for excystation. Cells were then imaged live with 5 min intervals using Nomarski optics and a ×60 oil objective (1.4 NA). To assess the effect of compound C-1 on egress using live microscopy, 16-well polystyrene dishes (Greiner Bio-One™) were similarly coated with 50 µg/mL fibronectin (Corning, catalog# 354008). HCT-8 cells were grown to > 95% confluence before infection with 15 × 10⁴ oocysts triggered for excystation. After 3 h, cells were gently washed with warm complete media to remove oocyst shells before adding either compound or DMSO. Tiled 2 × 2 images were taken live at 20 min intervals using a ×40 dry objective (0.7 NA).

Jumani R.S., Hasan M.M., Stebbins E.E., Donnelly L., Miller P., Klopfer C., Bessoff K., Teixeira J.E., Love M.S., McNamara C.W, & Huston C.D. (2019). A suite of phenotypic assays to ensure pipeline diversity when prioritizing drug-like Cryptosporidium growth inhibitors. Nature Communications, 10, 1862.

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Adhesion Assay for Bone Marrow Stromal Cells

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Forward primer (5'-3') Reverse primer (5'-3')
of bands were determined using Image J (version 1.52a). Band intensities of α-SMA and FAK were normalised to β-actin and p-FAK was normalised to FAK. Percentages of MC1 and control intensities were compared by paired Wilcoxon test.
Assay 10: adhesion assay BMSC adhesion to fibronectin-coated, collagen I-coated and uncoated surface was assessed. 96-well plates were coated overnight at 4 °C using 16 µg/ mL fibronectin or 0.75 mg/mL collagen I (Corning).
fibronectin-and collagen I-coated wells were blocked for non-specific binding with 1 % heat-inactivated BSA for 1 h at 37 °C. 2,500 cells/well were seeded in sextuplets. After 15 min, 30 min and 4 h, cell suspension was removed and non-adherent cells were washed away with PBS. Adherent cells were fixed using 4 % neutral buffered formalin and stained with Hoechst 33342 (ThermoFisher Scientific). 4 images per well were taken at predefined spots using a Nikon Eclipse Ti2 upright brightfield microscope. Cells were counted manually using ImageJ. Cell counts at 15 min and 30 min were normalised to the respective 4 h count. Percentage increase of adherent MC1 and control BMSCs between 15 min (settling time) and 30 min were calculated and compared using a paired t-test.

Heggli I., Epprecht S., Juengel A., Schuepbach R., Farshad-Amacker N., German C., Mengis T., Herger N., Straumann L., Baumgartner S., Betz M., Spirig J.M., Wanivenhaus F., Ulrich N., Bellut D., Brunner F., Farshad M., Distler O, & Dudli S. (2021). Pro-fibrotic phenotype of bone marrow stromal cells in Modic type 1 changes. European cells & materials, 41.

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Inhibition of Hypusination Pathway in Cell Culture

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Ciclopirox olamine (CPX), an iron chelator that inhibits the
hypusination enzyme deoxyhypusine hydroxylase was obtained from Santa Cruz
Biotechnology (Dallas, TX) and diluted in dimethyl sulfoxide (DMSO).
N1-guanyl-1,7-diamineheptane (GC7), a spermidine analog that inhibits the
activity of the hypusination enzyme deoxyhypusine synthase, was obtained from
Biosearch Technologies (Petaluma, CA) and diluted in sterile water. Recombinant
human TGFβ1 was diluted in 0.1% BSA (Thermo Fisher Scientific, Waltham,
MA) and used at a working concentration of 2.5 ng/mL. Control treatments used
DMSO (in place of CPX), sterile water (in place of GC7) or 0.1% BSA (in place of
TGFβ). Fibronectin (Corning, Bedford, MA) coating was performed by
treating plates with 3 μg/mL Fibronectin in sterile PBS at 37 °C
for 1 h, followed by one wash with sterile PBS prior to cell plating.

Güth R., Adamian Y., Geller C., Molnar J., Maddela J., Kutscher L., Bhakta K., Meade K., Kim S.L., Agajanian M, & Kelber J.A. (2019). DHPS-dependent hypusination of eIF5A1/2 is necessary for TGFβ/fibronectin-induced breast cancer metastasis and associates with prognostically unfavorable genomic alterations in TP53. Biochemical and biophysical research communications, 519(4), 838-845.

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Ostracod Epidermal Cell Culture

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Using optimal conditions for dissociation of the epidermal cells (incubation in Dispase I in M199 (0.6–1.0 U/ml) for 10 min), ostracod epidermal cells were plated on different substrates (i.e. cells were pooled from 30 ostracods per substrate-coated well) to promote adherence during culture in the optimised M199 medium described above. Substrates included collagen type I (48-well; Sigma), collagen type IV (48-well; Sigma), laminin (24-well; Corning), fibronectin (24-well, BioCoat; Corning), Matrigel (48-well; Sigma) and chitosan. For chitosan coatings, 4% (w/v) chitosan (>90% degree deacetylation; Sigma) in 2% (v/v) acetic acid was autoclaved, applied to 48-well plates and allowed to dry for 24 h to form a thin film. The acidity of the films was neutralised with 0.5 M NaOH and washed repeatedly with Hank’s balanced salt solution until film pH returned to a physiological range (pH = 7.4).

Morgan S.R., Paletto L., Rumney B., Malik F.T., White N., Lewis P.N., Parker A.R., Holden S., Meek K.M, & Albon J. (2020). Establishment of long-term ostracod epidermal culture. In Vitro Cellular & Developmental Biology. Animal, 56(9), 760-772.

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Hydrogel Formation and Characterization

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The hydrogel monomers, 4-arm PEG4norb, were purchased from JenKem Technology USA (Plano, TX, USA). The photoinitiator LAP was synthetized as described previously [52 ]. The fluorescent dye Alexa Fluor488 was obtained from Invitrogen (Waltham, MA, USA), and propidium iodide from Carl Roth GmbH (Karlsruhe, Germany). Trypsinized bovine collagen type I, PureCol, was obtained from Advanced BioMatrix (Carlsbad, CA, USA), and fibronectin from Corning GmbH (Wiesbaden, Germany). Purified water was filtered with MilliQ system, Merck Millipore, Burlington, MA, USA. TGFα, insulin and BPE (bovine pituitary extract) were supplied as parts of the DermaLife K LifeFactors kit from Lifeline Cell Technology (Frederick, MD, USA). Human recombinant EGF (E. coli-derived) was obtained from PromoCell (Heidelberg, Germany), and recombinant human TGFβ-1 (CHO-derived) was purchased from Peprotech (Hamburg, Germany). EGF receptor monoclonal antibody 225 was obtained from Life Technologies (Carlsbad, CA, USA). All other reagents; i.e., PEG-dithiol; FITC-dextran, 40 kDa; 10xDMEM; 7.5% NaHCO₃; fluorescein diacetate; mitomycin C; and EGFR inhibitor AG-1478, were obtained from Sigma-Aldrich (St. Louis, MO, USA).

Tomasova L., Guttenberg Z., Hoffmann B, & Merkel R. (2019). Advanced 2D/3D cell migration assay for faster evaluation of chemotaxis of slow-moving cells. PLoS ONE, 14(7), e0219708.

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Fluorescence Staining of Adherent Cells

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For fluorescence staining, respective cell lines were seeded on 50 µg/ml fibronectin (Corning) coated 8-well chamber slides (Ibidi) at a density of 12,000 cells per well. Cells were cultured in chamber slides under standard culture conditions (as described above) for 24 h. Thereafter, cells were fixed in 4% PFA (Electron Microscopy Sciences) in DPBS (Thermo Fisher Scientific) for 20 min. After fixation, samples were 3 times washed using DPBS and permeabilized by 0.1% Triton X-100 (Sigma Aldrich) in DPBS for 3 min. Thereafter, cells were stained for F-actin using fluorophore labeled Phalloidin (Thermo Fisher Scientific) and dsDNA by Hoechst 33342 (Thermo Fisher Scientific) in a permeabilization buffer for 2 h. Finally, samples were washed 5 times in DPBS and imaged in DPBS.

Bernhard P., Feilen T., Rogg M., Fröhlich K., Cosenza-Contreras M., Hause F., Schell C, & Schilling O. (2022). Proteome alterations during clonal isolation of established human pancreatic cancer cell lines. Cellular and Molecular Life Sciences, 79(11), 561.

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Esomeprazole-Induced Cell Migration

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Cells in T75 flasks were incubated for 72 hours with esomeprazole at the approximate LD50 dose, and subsequently underwent a 3 hour starving period using serum free medium. They were plated onto the upper chamber of a 24-well Boyden chamber coated with collagen type I and/or fibronectin (Corning B.V. Life Sciences, Amsterdam, The Netherlands; Cat. No. 3428) with an 8-?m pore polycarbonate membrane in medium without serum, and medium containing 10% fetal bovine serum was filled in the lower chamber as chemoattractant. After 12 hours, cells that did not migrate through the pores were removed using cotton swabs. Membranes were stained using crystal violet, and migrating cells were counted in 9 gridded high-power fields per membrane under an inverted microscope. One experiment was performed with 3 technical replicates, and confirmed with another independent experiment.

Lindner K., Borchardt C., Schöpp M., Bürgers A., Stock C., Hussey D.J., Haier J, & Hummel R. (2014). Proton pump inhibitors (PPIs) impact on tumour cell survival, metastatic potential and chemotherapy resistance, and affect expression of resistance-relevant miRNAs in esophageal cancer. Journal of Experimental & Clinical Cancer Research : CR, 33(1), 73.

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