The iPS cells generated by co-incubated with cordycepin (long-term) were named iPS-CN cells. The iPS-CN cells were transferred to Ultra-Low attached culture dishes and incubated in DMEM/High glucose containing 15% FBS, non-essential amino acids (0.1 mM), L-glutamine (2 mM), β-mercaptoethanol (0.1 mM) to generate embryoid bodies. After 4 days, the embryoid bodies were transferred to regular cell culture dishes containing ITS-FN medium (DMEM/F12 (GIBCO BRL), L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), 1% ITS-G media supplement (GIBCO BRL) and 5 μg/ml Fibronectin (GIBCO BRL)) for neural stem/precursor cell selection (4 days). After selection, the cells were transferred to poly-L-ornithine (GIBCO BRL)/Fibronectin-coated dishes containing medium with N-2 medium (DMEM/F12, L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml), 1% N-2 media supplement (GIBCO BRL)) supplied with basic fibroblast growth factor (bFGF; GIBCO BRL) for neural stem/precursor cell expansion. The final neural stem/precursor cells were collected for the further analysis.
Fibronectin
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, Australia, Canada
Fibronectin is a glycoprotein that plays a crucial role in cell adhesion, growth, migration, and differentiation. It is a component of the extracellular matrix and is involved in various biological processes. Fibronectin facilitates the attachment of cells to the extracellular matrix, providing a structural and functional support for cells.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (22 mentions)
Fibronectin, by Merck Group (15 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (14 mentions)
Laminin, by Thermo Fisher Scientific (14 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (11 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (11 mentions)
L glutamine, by Thermo Fisher Scientific (10 mentions)
Penicillin, by Thermo Fisher Scientific (9 mentions)
Streptomycin, by Thermo Fisher Scientific (8 mentions)
Dmem f12, by Thermo Fisher Scientific (8 mentions)
Trypsin edta, by Thermo Fisher Scientific (8 mentions)
Collagen 1, by Thermo Fisher Scientific (7 mentions)
Laminin, by Merck Group (7 mentions)
Matrigel, by Corning (7 mentions)
Poly l ornithine, by Merck Group (7 mentions)
α sma, by Thermo Fisher Scientific (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Matrigel, by BD (6 mentions)
Basic fibroblast growth factor (bfgf), by R&D Systems (6 mentions)
B27 supplement, by Thermo Fisher Scientific (6 mentions)
342 protocols using fibronectin
1
Cordycepin-Induced Neural Stem Cells
2
Fibroblast Dynamics on Micropillars
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NIH3T3 fibroblasts stably expressing H2B-EGFP were cultured in lowglucose Dulbecco's Modified Eagle Medium (Gibco; LifeTechnologies) supplemented with 10% (vol/vol) FBS (Gibco; Life Technologies) and 1% penicillin-streptomycin (Gibco; Life Technologies) at 37 °C and 5% CO2 in humid conditions. Cells were transfected with RFP-Lifeact or mcherry-MKL by electroporation (Gibco; Life Technologies), the day before the experiment. Cells were trypsinized (Gibco; Life Technologies) and seeded on micropillars coated with 20 µg/ml of Bovine Serum Albinum (BSA, Sigma Aldrich) and Fibronectin (Gibco; Life Technologies) for 3 h followed by 100 µg/ml of Fibronectin for 1 h. Before imaging, the chip was inverted in a petri dish, on two parafilm spacers to avoid contact between the cells and the bottom of the dish. A special medium (Gibco; Life Technologies, catalogue number: 18045), not requiring a CO2-rich atmosphere, was used during the experiments.
3
Micropatterning Cells with Dynamic Confinement
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Micropatterning was performed according to the manufacturer protocol (4Dcell, France). Briefly, coverslips were first coated with non-adhesive PLL-g-PEG. Lines were patterned on coverslips using a photomask (cat no. UM006; 4Dcell) and treatment with deep UV. The coverslips were then incubated with fibronectin (10 μg/ml) (cat no. 33016015; Thermo Fisher) for 1 hr at room temperature. To mark the adhesive patterns, a small amount of red fluorescent BSA (1 μg/mL) (cat no. A13101; Thermo Fisher) was added to the fibronectin solution. Cells were confined down to 3 μm using a dynamic cell confiner according to the manufacturer protocol (4Dcell). Because cells were found to be less motile in the dynamic cell confiner, all cells were treated with siRNA towards MYPT1 to generally increase rates of motility.
4
Decellularized ECM Substrate Preparation
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Decellularized ECM substrates were prepared by a previously reported method [9 (link)]. Briefly, CMSC29, DMSC23, or 3T3 cells were plated at a density of 1 x 103 cells/cm2 with media change performed every 2–3 days for up to 14 days. From day 3 onwards, 50 μM ascorbic acid (Wako, Japan) was added into the culture medium to increase the production of ECM. To decellularize the deposited ECM, the cultures were washed twice with PBS (Life Technologies) and treated with PBS containing 0.5% Triton X-100 (Thermo Scientific) and 20mM NH4OH (Sigma-Aldrich) for 5 min at 37°C. The ECM was washed with PBS, air-dried in the sterile biosafety cabinet and stored at 4°C for up to 1 month. dECM deposited by CMSC29, DMSC23, and 3T3 cells were designated as dECM-CMSC29, dECM-DMSC23, and dECM-3T3 respectively. Fibronectin-coated plates were prepared by incubating tissue culture plates with a 10 μg/mL Fibronectin (Life Technologies) diluted in PBS for at least 1 h at 37°C. Excess solution was removed and plates were washed twice with PBS.
5
Microcontact Printing of Fibronectin Patterns
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Microcontact printing was used to produce fibronectin-coated ring-shaped lanes. PDMS stamps were activated with UV light (PSD-UV, novascan) for 5 min. Then, the stamps were incubated for 45 min in a solution containing 40 μg/ml fibronectin (Yo proteins) and 10 μg/ml fibronectin labeled with Alexa Fluor 488 (Life Technologies) dissolved in ultrapure water. Next, stamps were washed with ultrapure water, dried and placed on a petri dish (μ-Dish, Ibidi), which had been activated with UV light for 15 min. A droplet of a 1 mg/ml poly-L-lysine-grafted polyethylene glycol (PLL-PEG) (2 kDa PEG chains, SuSoS) solution (dissolved in 10 mM HEPES containing 150 mM NaCl was placed at the edge of the stamps and drawn into the spaces between surface and stamp by capillary action. Stamps were removed and a glass coverslip was placed on the dish surface to ensure complete coverage of the surface with PEG solution. After a 30-min incubation, the coverslip was removed and the surface was washed three times with phosphate-buffered saline (PBS) and stored in PBS until cells were seeded. An area of up to 1.5 cm2 was patterned, resulting in up to 5000 ring patterns per dish.
6
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.
7
Culturing Insulin-Producing EndoC-βH1 Cells
8
Immunofluorescence Analysis of Heparanase and RNA Pol II
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The HRECs were seeded on glass coverslips precoated with 5 mg/ml of fibronectin (Gibco) and allowed to grow to semiconfluence in a culture dish. The cells were washed with phosphate buffered saline (PBS) three times and fixed in fresh 4% paraformaldehyde (pH 7–8) for 10 min at room temperature. PBS was composed of 2.89 g of Na2HPO4·12H2O, 8 g of NaCl, 0.2 g of KCl, 0.2 g KH2PO4 and 80 ml of ddH2O. Next, the HRECs were permeabilized in 0.1% of Triton X-100 (Sigma, St. Louis, MO) for 5 min and blocked with 1% albumin from bovine serum (BSA; Sigma) in PBS containing 0.1% Tween 20 (blocking solution) for 60 min at room temperature. The cells were then incubated with rabbit anti-human heparanase antibody (1:300 dilution; Abcam, Cambridge, MA) and mouse anti-human RNA Pol II antibody (1:200 dilution; Abcam) overnight at 4 °C for the expression of heparanase and RNA Pol II, followed by incubation with appropriate secondary antibodies conjugated with Alexa Fluor 488 and Alexa Fluor 555 (1:200 dilution; Boster Biologic Technology, Ltd., Wuhan, China) for 2 h at room temperature. The cells were stained with 100 ng/ml of 4',6-diamidino-2-phenylindole (DAPI; Sigma) for 5 min, mounted with an antifading fluorescence medium (Vector Laboratories, Burlingame, CA), and imaged using a laser scanning confocal microscope (Carl Zeiss, Jena, Germany).
9
Culturing Breast Cancer Cells on Fibronectin-Coated Substrates
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Human breast epithelial adenocarcinoma cells (MCF-7; ATCC) were cultured in minimum essential medium eagle’s (MEM) supplemented with 10% fetal bovine serum (FBS; ATCC), 0.1 mM non-essential amino acid (NEAA), 1 mM sodium pyruvate, and 1% antibiotic- antimycotic (anti-anti, Gibco). The cells were maintained in a humidified atmosphere of 5% CO2 at 37 °C. Before cell culture, the cover glass and P(CL-co-DLLA) (crosslinked and non-crosslinked) substrates were coated with 10 µg/mL of fibronectin (Sigma-Aldrich) for 1 h and excess fibronectin was washed by phosphate buffered saline (PBS; Gibco).
10
Transwell Cell Migration Assay
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Cell migration assays were performed using Millipore 24-well Millicell (Millipore) plates containing an 8-μm pore membrane. The bottom face of the membrane was coated with 10 μg/mL fibronectin (Gibco). Cells were harvested 24 h after transfection and suspended in FBS-free RPMI-1640 culture medium. The cells were then added to the upper chamber (2 × 104 cells/well), and 0.5 mL RPMI-1640 plus 20% FBS was added to the lower compartment. The Transwell-containing plates were incubated for 24 h in the incubator. After incubation, cells that had migrated to the lower surface of the filter membrane were fixed with 4% paraformaldehyde for 25 min at room temperature. The membrane was washed 3 times with PBS and stained with 0.1% crystal violet in methanol for 15 min at room temperature. Cells remaining on the upper surface of the filter membrane (non-migrating) were gently scraped off with a cotton swab. The lower surfaces (with migrating cells) were captured by photomicroscopy (BX51 Olympus, Japan), and the cells were counted blindly (five fields per chamber).
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