Type I collagen was not detected in conditioned press (Fig. conditioned press were aspirated and cells detached by 0.25% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA). The total quantity of cells and viability were determined by automated cell counter (Countess?; Life Systems, Carlsbad, CA, USA). Cells were lysed in CNTZ buffer (10?mM cacodylate-HCl, pH 6.0, 1.0?M NaCl, 0.01% (v/v) Triton X-100, 1?M ZnCl2 and 0.2?mg/ml NaN3). LDH activity This assay (Roche Diagnostics) was performed in 96-well plates (Nunc). Conditioned press (100?l) and reaction combination (100?l) was incubated for 30?min at ambient heat protected from light. Fifty microliters quit solution were added and the OD go through at 492?nm and 690?nm inside a microplate reader (Multiskan MCC/340; Labsystems, Helsinki, Finland). Collagen degradation Fragmented collagen in the cells and released into the press was measured as hydroxyproline colorimetrically (?gren et al., 2006). The amount of degraded collagen was indicated as g of hydroxyproline D-Glucose-6-phosphate disodium salt per explant. Type I collagen degradation and biosynthesis As an indication of type I collagen degradation, C-terminal telopeptide of type I collagen (ICTP) was measured by an enzyme immunoassay kit (Orion Diagnostica, Espoo, Finland). De novo synthesis of type I collagen was measured by type I C-terminal collagen propeptide (CICP) released into the conditioned medium (MicroVue; Quidel Corporation, San Diego, CA, USA). Also, day time-4 press were centrifuged at 15,000??for 15?min using 300?kDa cut-off products (Vivaspin 500; Sartorius, Epsom, UK) to isolate CICP from type I procollagen (Kopanska et al., 2013). Histology, MMP-1 immunohistochemistry and TUNEL immunohistofluorescence The fixed cells were inlayed in paraffin. Serial 5-m sections were slice from each block. Morphology was assessed in hematoxylin-eosin-stained sections. MMP-1 immunohistochemistry was performed with the EnVision Flex+ (K8000; Dako, Glostrup, Denmark) polymer peroxidase diaminobenzidine system (Skaland et al., 2010). Cells were 1st subjected D-Glucose-6-phosphate disodium salt to heat-induced epitope retrieval for 20?min at 97?C using Tris-EDTA solution pH 9.0 in the pre-treatment module (Dako). Subsequently, sections were incubated with the MMP-1 monoclonal antibody at 1:10 dilution (50?g/ml) for 2?h at ambient heat in the Dako Autostainer Link 48 and treated according to the manufacturer’s protocol. Selected sections that were not pre-treated were incubated with the MMP-1 antibody for 18?h at 4?C. Adjacent D-Glucose-6-phosphate disodium salt sections were incubated with bad isotype control at the same concentration. Sections were counterstained with hematoxylin and cover-slipped. Epidermal and stromal staining were scored separately by blinded older pathologist (L. H. C.) on a 4-tiered level: 0, no; +, poor; ++, moderate; +++, intense staining. TUNEL staining was carried out following pre-treatment with proteinase-K (20?g/ml) using the ApopTag? fluorescein in situ kit (Millipore). Images were captured using a fluorescence microscope (Eclipse Ti-U, Nikon, Amsterdam, Netherlands) equipped with a digital HSPA1 video camera (DS-Qi1Mc, Nikon). Cells extraction Tissues components were prepared for 18?h at 4?C using CNTZ buffer (20?l/mg tissue) optimized for collagenase extraction (Mirastschijski et al., 2002) and supplemented with EDTA-free proteinase inhibitor cocktail/1?M pepstatin (Roche Diagnostics, Mannheim, Germany). Cells extracts were kept at ?80?C until analyzed. Type I collagenolytic activity assay Enzymes were incubated with 0.25?g/ml type I collagen from bovine pores and skin (Millipore) with or without inhibitors/APMA as indicated in a total volume of 40?l with 25?M ZnCl2, and 1?mM CaCl2 in the presence of the proteinase inhibitor cocktail and pepstatin at 24?C for 240?h unless stated otherwise. Samples were electrophoresed on NuPAGE? 4C12% Bis-Tris gels (Existence Systems) under reducing conditions and gels stained with Colloidal Blue (Salsas-Escat et al., 2010). Gels were scanned and the degree of collagen digestion was calculated from your density of the 1, 2, 3/41 and 3/42 bands (Welgus et al., 1981) determined by ImageJ (National Institutes of Health, Bethesda, MD, USA) and indicated in percentage (%). MMP and TIMP analyses MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1, TIMP-2 and TIMP-4 were measured using Quantibody? human being MMP/TIMP array (RayBiotech, Norcross, GA, USA). MMP-1 and MMP-3 were quantified by ELISA packages (Boster Biological Technology, Fremont, CA, USA). MMP-2 material were estimated by gelatin zymography (Mirastschijski et al., 2002) relative to MMP-2 standard run in parallel using densitometry (ImageJ). TIMP-1 was assayed by ELISA kit from PeproTech (Henriksen et al., 2013). Western blot analyses Samples were electrophoresed using 10% sodium dodecyl sulfate-polyacrylamide gels under reducing conditions and electrotransferred onto polyvinylidene fluoride (PVDF) membrane (Immobilon? FL, Millipore) or nitrocellulose (Bio-Rad). The membranes were clogged with Odyssey buffer (Li-Cor, Lincoln, NE, USA) and incubated for 18?h at 4?C with main antibodies against MMP-1 diluted 1:500, MMP-3 diluted 1:250, type I collagen diluted 1:1,000 (nitrocellulose) and -actin diluted 1:70,000 (nitrocellulose). The membranes were incubated with coordinating.
Type I collagen was not detected in conditioned press (Fig
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