IGF-I, SB202190 (SB) and collagen I from rat tail were obtained from Sigma-Aldrich Inc. (Milan, Italy). PD98059 (PD) and 3-bromo-5-t-butyl-4-hydroxybenzylidenemalonitrile (AG1024) were purchased from Calbiochem (DBA, Milan, Italy). All compounds were solubilized in dimethylsulfoxide, except PD and IGF-I, which were dissolved in ethanol and in water, respectively. DDR1IN1 dihydrochloride (DDR-1 in) was purchased from Tocris Bioscience (Space, Milan, Italy).
Collagen 1 from rat tail
Manufactured by Merck Group
Sourced in Italy
Collagen I from rat tail is a natural, purified protein commonly used as a substrate for cell culture applications. It is extracted from the tail tendons of rats and provides a standardized, high-quality source of type I collagen, the most abundant form of collagen in the human body.
Automatically generated - may contain errors
Lab products found in correlation
N n methylenebisacrylamide, by Merck Group (2 mentions)
3 aminopropyltrimethoxysilane, by Merck Group (2 mentions)
N n n n tetramethylethylenediamine, by Merck Group (2 mentions)
Acrylamide monomer, by Merck Group (2 mentions)
Ammonium persulfate, by Merck Group (2 mentions)
N sulfosuccinimidyl 6 4 azido 2 nitrophenylamino hexanoate, by Merck Group (2 mentions)
Fibronectin from human plasma, by Merck Group (2 mentions)
Igf 1, by Merck Group (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Human fibronectin, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (1 mentions)
Laminin from human fibroblasts, by Merck Group (1 mentions)
Poly d lysine hydrobromide pdl, by Merck Group (1 mentions)
Glass bottom plates, by MatTek (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
6 protocols using collagen 1 from rat tail
1
Molecular Mechanisms of IGF-I Signaling
2
Collagen I Coating Protocol for Cell Culture
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Collagen I from rat tail (Sigma-Aldrich) was diluted to 50 μg·mL–1 in 200 mM acetic acid (Sigma) and added to each well
of the cell culture plate at 5 μg collagen·cm–2. The plates were incubated with the dissolved collagen for 1 h,
and after this incubation period, the collagen solution was removed,
and the plates were rinsed three times with 200 μL of PBS to
remove any residual acetic acid solution. The collagen-treated plates
were allowed to dry for 1 h in a laminar flow hood and stored for
less than 1 week at 4 °C prior to use.
of the cell culture plate at 5 μg collagen·cm–2. The plates were incubated with the dissolved collagen for 1 h,
and after this incubation period, the collagen solution was removed,
and the plates were rinsed three times with 200 μL of PBS to
remove any residual acetic acid solution. The collagen-treated plates
were allowed to dry for 1 h in a laminar flow hood and stored for
less than 1 week at 4 °C prior to use.
3
Polyacrylamide Gel Fabrication and ECM Functionalization
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To prepare polyacrylamide gels, glass coverslips were washed twice with 70% ethanol, activated with 0.1 m NaOH, and silanized with (3‐aminopropyl)trimethoxysilane (Sigma). The glass coverslips were then treated with 0.5% glutaraldehyde (Sigma) for 30 min and washed extensively with sterile Milli‐Q water. Mixtures of Milli‐Q water, acrylamide monomers (Sigma), and crosslinker N,N‐methylene‐bis‐acrylamide (Sigma) were prepared according to previously determined formulations to obtain 2 kPa gels.[49 ] For the polymerization reaction, 5 µL of 10% ammonium persulfate (Sigma) and 0.75 µL N,N,N′,N′‐tetramethylethylenediamine (Sigma) were added into 0.5 mL mixtures. To functionalize gels with ECM proteins, gels were first treated with 1 mg mL−1N‐sulfosuccinimidyl‐6‐(4′‐azido‐2′‐nitrophenylamino) hexanoate (Sigma), which was activated by ultraviolet (UV) light. Finally, gels or silanized glass coverslips were incubated with 10 µg mL−1 collagen I from rat tail (Sigma) or human fibronectin (Sigma) for 3 h at room temperature (RT) and UV‐sterilized prior to cell seeding.
4
Sucrose and Lactose Effects on Cell Adhesion
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Cells were incubated for 24 h in DMEM/10%FBS/1%PenStrep media supplemented with no additional sugar, 50 mM sucrose or 50 mM lactose. Tissue culture grade 24-well plates were coated overnight with an ECM model mixture containing 0.5 μg/cm2 laminin from human fibroblasts (Sigma), 2.5 μg/cm2 fibronectin from human plasma (Sigma), and 6 μg/cm2 collagen I from rat tail (Sigma). Wells were blocked for 30 min with 1% BSA to block nonspecific binding and washed 3× with serum-free DMEM prior to addition of cells. Cells were harvested using 1 mM EDTA, washed 2× with serum-free DMEM and incubated with or without 10 mM EDTA (Thermo), to inhibit integrin activity, for 30 min in serum-free DMEM. Cells were added in the presence or absence of 10 mM EDTA in serum-free DMEM with no additional sugar, 10 mM sucrose or 10 mM lactose to ECM coated plates and incubated for 60 min at 37 °C. At end of incubation plates were placed on ice and processed for western blotting as described below.
5
2D Cell Migration Substrate Preparation
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For 2D cell migration experiments, cells were plated onto substrates coated with collagen I from rat tail (Sigma Aldrich, St. Louis), fibronectin from human plasma (Sigma Aldrich), or poly-d-lysine hydrobromide (PDL) (Sigma Aldrich). Collagen I and fibronectin were dissolved in PBS, and PDL was dissolved in sterile milliQ water. Twenty-four well glass bottom plates (MatTek, Ashland, MA) were coated with 300 µl (per well) of 20 μg/ml of the varying protein solutions and incubated for 1 h at 37°C. Following incubation, wells with collagen and fibronectin were washed 3 times with PBS, while wells with PDL were washed 3 times with sterile milliQ water. After washing the substrates, 1 × 104 cells were plated into each well. Cells were imaged and analyzed as described below.
6
Functionalized Polyacrylamide Gel Fabrication
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To prepare polyacrylamide gels, glass coverslips were washed twice with 70% ethanol, activated with 0.1 M NaOH, and silanized with (3-aminopropyl) trimethoxysilane (Sigma). Then the glass coverslips were treated with 0.5% glutaraldehyde (Sigma) for 30 min and washed extensively with MilliQ water. Mixtures of MilliQ water, acrylamide monomers (Sigma), and crosslinker N,N-methylene-bis-acrylamide (Sigma) were prepared according to previously determined formulations62 ,63 (link). For the polymerization reaction, 5 μl of 10% ammonium persulfate (Sigma) and 0.75 μl N,N,N′,N′-tetramethylethylenediamine (Sigma) were added into 0.5 ml mixtures. To functionalize gels with collagen, gels were first treated with 1 mg/ml N-sulfosuccinimidyl-6-(4′-azido-2′-nitrophenylamino) hexanoate (Sigma), which was activated by ultraviolet (UV) light. Finally, gels were incubated with 10 μg/ml collagen I from rat tail (Sigma) or human collagen VI (Rockland) for 3 h at room temperature (RT) and UV-sterilized prior to cell seeding.
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