DU145 cells were grown for 2 days on coverslips that were layered with 100% matrigel (Corning) and DQ-collagen IV (Invitrogen Life Technologies, diluted at final concentration of 25 μg/mL). After treatment overnight with drug in presence or absence of HGF, cells were fixed in 4% (w/v) paraformaldehyde for 30 minutes. Next, cells were washed 2 times with PBS and incubated with Alexa Fluor 635 phalloidin (Life Technologies) diluted 1:200 in BSP for 30 minutes to stain the actin cytoskeleton. Cells were then washed 2 times with PBS and coverslips were mounted. Colonies and cleaved DQ-collagen IV were visualized on Leica TCS SP5 Laser scanning confocal microscope and images were captured using LAS AF software.
Dq collagen 4
Manufactured by Thermo Fisher Scientific
Sourced in United States
DQ-collagen IV is a fluorescent-labeled collagen IV product used for the detection and quantification of collagen IV in biological samples. It is a useful tool for researchers studying extracellular matrix components and their role in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Matrigel, by Corning (2 mentions)
Alexa fluor 635 phalloidin, by Thermo Fisher Scientific (1 mentions)
Af6000 lx microscope, by Leica (1 mentions)
Matrigel matrix, by Corning (1 mentions)
35 mm glass bottom culture dishes, by MatTek (1 mentions)
Celltracker deep red dye, by Thermo Fisher Scientific (1 mentions)
Image pro plus, by Media Cybernetics (1 mentions)
Facscalibur, by BD (1 mentions)
Propidium iodide, by BD (1 mentions)
Soybean trypsin inhibitor, by Merck Group (1 mentions)
Spectramax m2 microplate reader, by Molecular Devices (1 mentions)
Cls3925, by Corning (1 mentions)
Matrigel, by BD (1 mentions)
Phalloidin 633, by Thermo Fisher Scientific (1 mentions)
Anti gfp jl 8, by Takara Bio (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ab101349, by Abcam (1 mentions)
Ab179824, by Abcam (1 mentions)
Anti β actin ac 15, by Merck Group (1 mentions)
Matrigel growth factor reduced basement membrane matrix, by Corning (1 mentions)
12 protocols using dq collagen 4
1
Analyzing Actin Cytoskeleton and Collagen IV Cleavage
2
Quantifying Cell Migration and Invasion
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Cell migration was assessed using time-lapse microscopy as previously reported [30 ] with slight modifications. In brief, approximately 2 × 104 cells in each study group were seeded in 6-well plates in their corresponding pHe medium. On the next day, cells were placed on a Leica AF6000 LX microscope and their movement was recorded for 3 h at 20 min intervals at 37 °C. Individual cells were tracked for motility analysis with Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). To determine cell invasive capability, cells were stained with 1 μM CellTracker Deep Red dye (Invitrogen), and then seeded in 35-mm glass bottom culture dishes (MatTek, Ashland, MA, USA) precoated with Matrigel Matrix (Corning, Tewksbury, MA, USA) supplemented with 25 μg/mL DQ-collagen IV (Invitrogen). The dishes were left for 16-18 h before nuclear staining with 1 μg/mL Hoechst 33342. Images of live-cell invasion were photographed by the TCS SP5 confocal microscope followed by quantitative analysis using Imaris 8.0 software.
3
Intracellular DQ-Collagen IV Degradation
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The effect of fusion proteins and of combinations of proteins on intracellular DQ-collagen IV degradation was assessed according to the protocol described in Mirković et al., [60 (link)]. 5 × 104 MCF10A neoT cells were plated into the wells of a 24-well plate. Cells were allowed to attach overnight, when the media were replaced with serum free medium containing 10 μM E64d, 0.63 μM of recombinant fusions or combinations of proteins. After 4 h incubation, DQ-collagen IV (5 μg/ml, Invitrogen) was added and the cells were incubated for an additional 2 h. Propidium iodide (BD Biosciences, CA, USA) exclusion was used to monitor just the viable cells. The measurements were performed on a BD FACS Calibur instrument (Beckton Dickinson, Inc.), analysis using FlowJo software (FlowJO, Ashland, OR, USA).
4
Quantification and Visualization of DQ-Collagen IV Degradation
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Quantification of intracellular and extracellular DQ-collagen IV (Invitrogen) degradation as well as its visualization were performed as reported [19 (link)].
5
Matrigel Invasion Assay for MMP Activity
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Twenty four-well Matrigel invasion chambers were incubated with DQ-collagen IV (Invitrogen, Life Technologies) diluted in serum-free DMEM at a final concentration of 20 ng/ml at 37 °C in dark overnight. The chambers were briefly rinsed with serum-free DMEM. 1 × 104cells were seeded into each invasion chamber in the presence or absence of pcDNA-ERβ and siRNA-ERβ. Cells were then incubated in a CO2 incubator at 37 °C for 4 h. Proteolytic degradation of DQ-collagen IV in the BM was imaged with fluorescence microscopy. When DQ-collagen IV was de-gradated by the MMPs secrete by cells, the clipped DQ-collagen may show fluorescence. A brighter fluorescence present the higher invasion ability. A study by Chen H et al. also described the protocol [27 (link)].
6
Quantifying Collagen Degradation in Cells
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LabTek 4-well chamber slides were coated with phenol-red free basement membrane matrix, containing 30 ng/ml DQ-Collagen IV (Invitrogen Life Technologies, Carlsbad, CA, USA). After 12 min solidification, 3×104 cells in phenol-red free RPMI with 1% FCS were plated on top of the coated basement membrane matrix. After 48 h incubation, cells were fixed with 2% PFA, stained with Hoechst and mounted. Degradation products of the DQ-substrate (green fluorescence) were analyzed on an automated microscope (scan∧R - Olympus) using a 20x, N.A. 0.75 UPLSAPO objective with Hoechst emission measured at 440–475 nm and DQ-Collagen at 520–550 nm. The mean intensity of DQ-substrate degradation was calculated using the scan∧R Analysis Software (v. 1.2.0.6) and normalized to the number of cell nuclei.
7
Activation and Kinetic Analysis of MMP3
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All MMP3-D4K-4 and MMP3-D4K-4 fusion protein samples were exchanged into cleavage buffer (10 mM Hepes, 150 mM NaCl, and 10 mM CaCl2 at pH 7.5). MMP3-D4K-4 was activated with 16 units of EK (NEB, P8070L) for every 25 μg protein through incubation at room temperature for 16 h. To inactivate the EK, 0.1 mg/ml soybean trypsin inhibitor (Sigma, 17075029) was added to the protein solution. Fifty microliter of 2.5 μM fluorogenic MMP3 peptide substrate (R&D Systems, ES002) or 50 μg/ml DQ-collagen-IV (Invitrogen, D12052) in cleavage buffer was combined with the desired concentration of activated MMP3-D4K-4 sample within a 96-well black flat-bottom plate (Corning, CLS3925), and the fluorescence signal was measured on a Molecular Devices SpectraMax M2 microplate reader (Molecular Devices) with 320/405 nm and 485/535 nm excitation/emission for the peptide substrate and DQ-collagen-IV, respectively. Concentration-dependent assays were performed in duplicate with 2.5-fold dilutions from 400 nM. Curve fits were generated in GraphPad Prism using a nonlinear dose–response model with variable slope and four parameters.
8
3D Culture of Cells with DQ-collagen IV
9
Fibrotic Lung Fibroblast Invasion Assay
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Six-well Matrigel invasion chambers were incubated with DQ-collagen IV (Molecular Probes, Eugene, OR) diluted in serum-free DMEM at a final concentration of 25 μg ml−1 at 37 °C in dark overnight. The chambers were briefly rinsed with serum-free DMEM. Fibrotic lung fibroblasts were trypsinized from stiff matrix. In all, 1 × 105 cells were seeded into each invasion chamber in the presence or absence of NKI-GoH3 (10 μg ml−1), α6-siRNA and MMP-2/MMP-9 Inhibitor I (25 μM). Cells were then incubated in a CO2 incubator at 37 °C for 3 h. Proteolytic degradation of DQ-collagen IV in the BM was imaged with confocal laser-scanning microscopy as described below.
10
3D Collagen IV Degradation Assay
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3D culturing was performed as previously described with slight modifications [27 , 52 (link)]. Matrigel (BD Bioscience) supplemented with 25 μg/mL DQ-Collagen IV (Molecular Probes) was applied to glass coverslips and allowed to solidify at 37°C for 30 minutes. 1×105 cells were seeded on top of the 3D matrix in 2 mL complete media. Cells were allowed to settle for 2 days prior to treatment with either 100 ng/mL EGF or 33 ng/mL HGF for 2 additional days. To terminate the experiment, the culture was fixed for 30 minutes in warm 4% PFA at 37°C. Actin was then stained using 1:200 Phalloidin 633 (Molecular Probes) for 30 minutes in warm BSP at 37°C. Three fields from three independent experiments were imaged and representative images are shown. To quantify cleaved DQ-Collagen IV signal, the area of the actin cytoskeleton was subtracted from the DQ-Collagen IV fluorescent signal using Image Calculator (ImageJ). Remaining extracellular DQ-Collagen IV fluorescence was assessed by integrated density (ImageJ) and displayed as arbitrary units.
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