Plasma bovine fibronectin (5 μg, Sigma), human cellular fibronectin (5 μg, Sigma) and rat and human fibronectin aggregates (5 μg, see above) were respectively incubated with 100 ng recombinant human active MMP3 (Abcam, Cambridge, UK), MMP7 (Millipore, Darmstadt, Germany), and MMP9 (Millipore, San Diego, CA) in 50 µl MMP‐reaction buffer (50 mM Tris‐HCl, 0.15 M NaCl, 5 mM CaCl2, 0.05% Brij‐35, pH 7.5) at 37°C for 24 hr. For Western blot analysis, the reaction was terminated by adding non‐reducing SDS sample buffer. For coating purposes, the enzymatic activity of MMP7 was terminated by heating at 95°C for 10 min. Alternatively, fibronectin structural variants were incubated with PBS or cell conditioned media (35 µl) in the presence or absence of general MMP‐activator, 4‐aminophenylmercuric acetate (APMA, 2 mM, Sigma) in 50 μl MMP reaction buffer and incubated at 37°C for 72 hr.
Human cellular fibronectin
Manufactured by Merck Group
Sourced in United States
Human cellular fibronectin is a purified extracellular matrix protein derived from human cells. It is a high-molecular-weight glycoprotein that plays a key role in cell adhesion, growth, migration, and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Mmp 9, by Merck Group (1 mentions)
Mmp 7, by Merck Group (1 mentions)
Prism 5, by GraphPad (1 mentions)
Human plasma fibronectin, by Merck Group (1 mentions)
Sonorex super rk 102 h, by Bandelin (1 mentions)
Eclipse e400, by Nikon (1 mentions)
Matrigel, by BD (1 mentions)
Transwell cell culture chamber, by Corning (1 mentions)
Matrigel, by Nikon (1 mentions)
4 protocols using human cellular fibronectin
1
Fibronectin Cleavage by MMPs
2
Adhesion and Proliferation of PANC-1 Cells
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96-well plates were coated with 20 μg/ml human collagen IV and with 10 μg/ml human cellular fibronectin and human plasma fibronectin (Sigma Aldrich) for 2 h at 37 °C and then washed three times with PBS. PANC-1 cells were plated at a concentration of 1 × 105 cells/well in a 96-well plate for 30 minutes at 37 °C with different concentrations of NT4 (from 1 μM to 10 μM). Cells were then fixed with PBS - 4% PFA for 15 minutes at room temperature and stained with 0.1% crystal violet in 200 mM MES (2-(N-morpholino)ethanesulfonic acid) pH 6.0 for 1 h at room temperature. The cells were then solubilized with 10% acetic acid and the absorbance was measured at 595 nm using a microplate reader. The experiment was performed twice in triplicate.
EC50 values were calculated by non-linear regression analysis using Graph Pad Prism 5.03 software.
EC50 values were calculated by non-linear regression analysis using Graph Pad Prism 5.03 software.
3
Micropatterning Cells on Functionalized Coverslips
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20 × 20 mm or 24 × 24 mm coverslips (Carl Roth, Karlsruhe, Germany) were placed in a sonication bath (Sonorex Super RK102H, Bandelin, Berlin, Germany) in ethanol for 30 min and dried. The surfaces were passivated with 0.1 mg/ml poly-L-lysine-polyethylene-glycol (PLL-PEG, Surface solutions, Dübendorf, Switzerland) in HEPES solution before photo-micropatterning using a UV-ozone cleaner (Model 342-220, Jelight Company Inc., Irvine, CA, USA). The surfaces were subsequently equilibrated in PBS and incubated with 0.0625 g/cm human cellular fibronectin (Sigma Aldrich, St. Louis, MO, USA) in PBS for one hour before washing with PBS. For the experiments a circle- and crossbow-pattern was used, where every spot has a diameter of 50 µm, to ensure only one cell spreads on every micropattern.
4
Transwell Invasion Assay for LM38-LP Cells
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Transwell cell culture chambers (Corning, Union City, CA, USA) were used for the invasion assay. The filters (8 μm membrane pores) were first coated with a thin layer of Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's instructions. The lower chamber was supplemented with human cellular fibronectin (16 μg/ml) (Sigma-Aldrich, St. Louis, MO, USA) in 0.5 ml MEM, as chemo-attractant. 1x10 5 LM38-LP cells or its LEP/MEP components pre-treated or not with ATRA for 5 days, were seeded into the chambers in triplicate. After 24 h, the cells on the upper side of the membranes were thoroughly wiped off with a cotton swab, after which the membranes were fixed with formalin and stained with DAPI. The cells that had invaded the Matrigel, passed through the pores and re-attached to the lower surface of the filter were regarded as invasive, and their nuclei were counted in 400x fields under a fluorescence microscope (Eclipse E400, Nikon, Tokyo, Japan).
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