Mouse monoclonal anti-caveolin-1 and mouse monoclonal anti-Rac1 antibodies were from Transduction Laboratories (Lexington, KY, USA). Rabbit polyclonal anti-actin was from R&D Systems (Minneapolis, MN, USA). Mouse monoclonal anti-Rab5, Rabbit Polyclonal anti-AT1R, and AT2R antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-rabbit and goat anti-mouse immunoglobulin G (IgG) antibodies coupled to horseradish peroxidase were from Bio-Rad Laboratories (Hercules, CA, USA). Cell medium and antibiotics were from GIBCO Life Technologies (Grand Island, NY, USA). Fetal bovine serum (FBS) was from HyClone Laboratories (Logan, UT, USA). Glutathione-Sepharose 4B was from GE Healthcare (Piscataway, NJ, USA). The chemiluminescent substrate (EZ-ECL) and protein A/G beads were from Pierce Chemical (Rockford, IL, USA). The AT2R lentiviral short hairpin RNAs (shRNA) and the PTP1B inhibitor Cinn-gel-2ME were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human fibronectin was from Becton Dickinson (San Jose, CA, USA). The AT2R lentiviral expression vector was kindly provided by Prof. Walter G. Thomas (University of Queensland, Brisbane, Australia). The soluble alkaline phosphatase substrate p-Nitro phenyl Phosphate (pNPP) was from Santa Cruz, CA, USA.
Human fibronectin
Manufactured by BD
Sourced in United States, Germany
Human fibronectin is a glycoprotein found in the extracellular matrix and blood plasma. It plays a role in cell adhesion, migration, and wound healing. The core function of human fibronectin is to facilitate cell-cell and cell-matrix interactions.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Lhc basal medium, by Thermo Fisher Scientific (3 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
Rabbit polyclonal anti actin, by R&D Systems (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Human fibronectin, by Merck Group (2 mentions)
Polycarbonate filter, by GE Healthcare (2 mentions)
Matrigel, by BD (2 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (2 mentions)
Human collagen, by BD (2 mentions)
Stem pro accutase solution, by Thermo Fisher Scientific (2 mentions)
Complete egm 2 medium, by Lonza (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Horseradish peroxidase, by Bio-Rad (1 mentions)
Cell medium and antibiotics, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Fetal bovine serum (fbs), by Cytiva (1 mentions)
Alexa fluor 568 conjugated donkey anti rabbit, by Thermo Fisher Scientific (1 mentions)
Immunomagnetic separation system, by Miltenyi Biotec (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
51 protocols using human fibronectin
1
Detecting Caveolin-1, Rac1, and Rab5 Proteins
2
Immunofluorescence Imaging of STIM1 and TRPV4
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MDCK or HEK293 cells were seeded overnight at 60% confluence onto culture slides coated with human fibronectin (Becton Dickinson, MA). The cells were washed several times with ice-cold PBS and fixed in 3% paraformaldehyde for 10 minutes. The fixed cells were permeabilized with 0.1% Triton X-100 for 10 minutes and blocked for 1 hour in PBS containing 5% BSA (Sigma, USA) and 0.1% Tween. Following incubation with a polyclonal antibody against STIM1 or monoclonal antibody against TRPV4, the cells were washed and stained further with a conjugated donkey anti-rabbit IgG prior to processing the slides for immunofluorescence. After an additional 20 minutes of incubation at 37 °C, the cells were fixed, permeabilized, and decorated with either an anti- STIM1 or TRPV4 antibody. As a secondary antibody, Alexa Fluor 568-conjugated donkey anti-rabbit or Fluor 488-conjugated goat anti-mouse (Molecular Probes, Inc., Eugene, OR) was used. Confocal microscopy analysis was performed LSM710 (Zeiss, Germany) at the Center for Experimental Research Facilities of Chungbuk National University.
3
Expansion of hematopoietic cells from B. mori cocoons
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B. mori silkworm cocoons were supplied by Tajima Shoji Co., Ltd. (Yokohama, Japan). Pharmed tubing was from Cole-Parmer (Vernon Hills, IL, USA). The immunomagnetic separation system was from Miltenyi Biotech (Bergisch Gladbach, Germany and Bologna, Italy). Recombinant human TPO, interleukin-6 (IL-6), interleukin-11 (IL-11), human bone morphogenetic protein 4 (BMP4), human vascular endothelial growth factor (VEGF), human fibroblast growth factor (FGF), human Fms-related tyrosine kinase three ligand (Flt3L), human stem cell factor (SCF) were from Peprotech (London, UK). CHiR 99021 was from TOCRIS. TruCount tubes and human fibronectin were from Becton Dickinson (S. Jose, CA, USA). The following antibodies were used: mouse monoclonal anti-CD61, clone SZ21, from Immunotech (Marseille, France); rabbit monoclonal anti-β1-tubulin was a kind gift of Prof. Joseph Italiano (Brigham and Women's Hospital, Boston, USA). Alexa Fluor conjugated secondary antibodies and Hoechst 33258 were from Life Technologies (Monza, Italy). Additional details can be found in the ‘Key resources table’.
4
Antibody Characterization of Caveolin-1
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Rabbit polyclonal anti-CAV1, mouse monoclonal anti-CAV1, mouse monoclonal anti-pY14-CAV1, and mouse monoclonal anti-E-cadherin (BD Transduction Laboratories, Lexington, KY, USA), mouse monoclonal anti-PTPN14, and rabbit polyclonal anti-actin (R&D Systems, Minneapolis, MN, USA) antibodies were used as indicated by the manufacturers. Goat anti-rabbit and goat anti-mouse IgG antibodies coupled to horseradish peroxidase (HRP) were from Merck-Millipore (Billerica, Massachusetts, USA) and KPL Laboratories (Washington DC, USA), respectively. The ECL chemiluminescent substrate and the BCA protein determination kit were from Pierce (Rockford, IL, USA). The Plasmid Midi Kit was from Qiagen (Valencia, CA, USA). Human fibronectin was from Becton Dickinson (San Jose, CA, USA). Hygromycin was from Calbiochem (La Jolla, CA, USA). Fetal bovine serum (FBS) was from Biological Industries (Cromwell, CT, USA). Cell culture media and antibiotics were from GIBCO (Invitrogen, Carlsbad, CA, USA), Fugene 6® from Roche (Basel, Switzerland).
5
Culturing 16HBE14o- Bronchial Epithelial Cells
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The 16HBE14o- cell line [75 (link)], a human bronchial epithelial cell line, was kindly provided by Dieter Gruenert (passage number P2.54; University California, San Francisco, CA, USA). Cells (passage number 11) were maintained in minimum essential media (MEM) 1x medium (with Earle’s Salts, 25 mM HEPES and without L-glutamine; Gibco BRL), supplemented with 1 % L-glutamine, 1 % penicillin/streptomycin and 10 % foetal calf serum. For experimental cultures, cells were seeded at a density of 0.5 x 106 cells/insert on transparent BD Falcon cell culture inserts (surface area of 0.9 cm2, pores with 3.0 μm diameter, PET membranes for 12-well plates). The cell culture inserts were pretreated with 150 μL fibronectin coating solution, containing 0.1 mg/mL bovine serum albumin (Sigma-Aldrich), 1 % bovine collagen Type I (BD Biosciences) and 1 % human fibronectin (BD Biosciences) in LHC Basal Medium (Sigma-Aldrich). Inserts were placed in BD Falcon™ tissue culture plates (12-well plates) with 1 mL medium in the upper and 2 mL in the lower chamber. The cells were kept at 37 °C in 5 % CO2 humidified atmosphere for 7 days (medium changed after 3–4 days).
6
Glass Coverslip Cleaning and Passivation
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Thickness corrected glass coverslips (d=170±10 μm, Assistent, Karl Hecht KG, Sondheim, Germany) were cleaned by the following detergent treatment: ultrasonication in 2% Hellmanex solution (Hellma, Müllheim, Germany) for 10 min, flushing thoroughly with ultrapure water produced by a water purification system (Milli-Q Gradient A10, Millipore, San Francisco, CA) and again ultrasonication (2 × 10 min) in ultrapure water followed by repeated flushing with ultrapure water. To prevent unspecific interactions in GUV and RBC experiments, bare glass was passivated by incubation with 5 mg ml−1 bovine serum albumin (BSA, Sigma, Saint Louis, MO, USA) for 15 min. For macrophages, cell culture dishes (3-cm diameter, Greiner, Solingen, Germany) with thickness corrected glass coverslips were pre-coated with 10 μg ml−1 human fibronectin (BD Biosciences, Bedford, USA) in PBS for 30 min at 37 °C. In all cases, excess protein was removed by exchanging the buffer in a series of 10 washing steps.
7
Culturing Monkey Embryonic Stem Cells
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Monkey ESCs were cultured at 37°C and 5% CO2 in the MT-fCFA medium. For passaging, the cells were washed twice with phosphate-buffered saline (PBS) and were treated with 0.01% trypsin/0.004% EDTA (Sigma) for 2 min, followed by pipetting to ensure single-cell dissociation. The single dissociated cells were then mixed with 250 µg/ml of trypsin inhibitor (Gibco) to inactivate trypsin, centrifuged at 180×g for 5 min, and resuspended in the MT-fCFA medium containing 2.4 µM thiazovivin (Wako) and 4.7 µg/ml human fibronectin (BD Biosciences). The cells were plated on collagen type I-coated dishes (IWAKI) at a dilution ratio of 1∶8–1∶10. Cells routinely received fresh medium every day and were passaged when 80–90% confluence was reached, which normally occurred every 2–3 days.
For cryopreservation, single dissociated cells were suspended in STEM-CELLBANKER (ZENOAQ, BLC-3S) [17] (link) and directly frozen at −80°C without a programmed freezer in accordance with the manufacturer's instructions.
For cryopreservation, single dissociated cells were suspended in STEM-CELLBANKER (ZENOAQ, BLC-3S) [17] (link) and directly frozen at −80°C without a programmed freezer in accordance with the manufacturer's instructions.
8
Fibronectin-Mediated Cell Adhesion and Spreading
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For experiments using a fibronectin matrix, plastic dishes, multi-well cell culture plates or glass coverslips were coated with 20 μg/ml human fibronectin (BD Biosciences) at 4°C overnight. Before use, the coated cell culture ware was blocked with 1% bovine serum albumin (Carl Roth) for 1 h at room temperature and rinsed with 1 × PBS.
For re-plating assays, cells were detached using 0.5% Trypsin/EDTA (PAN-Biotech) and diluted in suspension media (normal media containing only 1% fetal bovine serum) to which 0.5 mg/ml soybean trypsin inhibitor type II (Sigma) was added. Cells were collected by centrifugation at 500 × g for 5 min, resuspended in fresh suspension media and then kept in suspension for 1 h at 37°C using a MacsMix Rotator (Miltenyi Biotec). After rotation, the cells were re-plated on fibronectin. Both the maintenance in 1% serum and the recovery period at 37°C were essential for adequate adhesion and spreading.
For re-plating assays, cells were detached using 0.5% Trypsin/EDTA (PAN-Biotech) and diluted in suspension media (normal media containing only 1% fetal bovine serum) to which 0.5 mg/ml soybean trypsin inhibitor type II (Sigma) was added. Cells were collected by centrifugation at 500 × g for 5 min, resuspended in fresh suspension media and then kept in suspension for 1 h at 37°C using a MacsMix Rotator (Miltenyi Biotec). After rotation, the cells were re-plated on fibronectin. Both the maintenance in 1% serum and the recovery period at 37°C were essential for adequate adhesion and spreading.
9
Fibronectin Cleavage Assay for DDR2
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ES2 or A2780 shRNAi-depleted of DDR2 or transduced with scrambled control (SCRM) were cultured for 6hours under serum free conditions on 2mg/mL of collagen I, and cell free, conditioned media was collected and incubated with 4ug human Fibronectin (BD). Extent of cleavage was measured at time intervals of 0.25, 1, and 16 hours by SDS Page followed by coomassie staining. Experiment was repeated 3 independent times. Amount of intact FN (~220 kDa) quantified by densitometry, and results of independent experiments plotted as mean +/− SEM. Control FN was incubated for 16hrs with non-conditioned media.
10
Quantifying Cell Migration and Invasion
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