To screen for MMP activity, an MMP array assay was conducted using a human MMP antibody array kit (Abcam, ab134004), targeting MMP-1, 2, 3, 8, 9, 10, and 13 and TIMP-1, 2, and 4. To quantify the concentrations of MMP-2 and MMP-9, human MMP-2 and MMP-9 ELISA kits (Raybiotech, ELH-MMP2-1, ELH-MMP9-1) were used. Samples were diluted 10 times before measurement.
Ab134004
Manufactured by Abcam
Sourced in United Kingdom
Ab134004 is a lab equipment product from Abcam. It is a recombinant monoclonal antibody.
Automatically generated - may contain errors
Lab products found in correlation
5 protocols using ab134004
1
Quantifying MMP Activity via ELISA
2
Quantification of MMP Levels in TNFα-Stimulated Chondrocytes
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Chondrocytes were stimulated with 10 ng/ml human TNFα at RT for 1 day and collected for human MMP analysis. The stimulated cells were lysed in 1X RIPA buffer including phosphatase and proteinase inhibitors and assessed according to the manufacturer's protocol (cat. no. ab134004; Abcam). To analyze the array data, comparison of signaling intensities for individual spots was detected using the UVItech system (Cleaver Scientific Ltd.) and analyzed with ImageJ 1.52a version software (National Institutes of Health).
3
Quantitative Proteomics of Cell Migration
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All cell lines underwent quantitative proteomics based on tandem mass tagging.
After embedding the cells in a 1.5-mg/mL collagen I matrix in an Ibidi µ-Slide Angiogenesis slide (Ibidi), the cells were allowed to move for 96 h under incubator conditions. The cleaved collagen was marked with an anti-Col1-3/4 C antibody (collagen type I cleavage site, Immunoglobe, Himmelstadt, Germany). For the analysis of matrix metalloproteinases (MMPs), the MMP array (ab134004, Abcam, Cambridge, UK) was applied.
After embedding the cells in a 1.5-mg/mL collagen I matrix in an Ibidi µ-Slide Angiogenesis slide (Ibidi), the cells were allowed to move for 96 h under incubator conditions. The cleaved collagen was marked with an anti-Col1-3/4 C antibody (collagen type I cleavage site, Immunoglobe, Himmelstadt, Germany). For the analysis of matrix metalloproteinases (MMPs), the MMP array (ab134004, Abcam, Cambridge, UK) was applied.
4
Modulation of Inflammatory Markers and ECM Components
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Conditioned medium from cancer tissue indirectly co-cultured with omentum and pre-treated with our various treatments (see material and methods above) was thawed at room temperature. Protein arrays for human growth factors and cytokines (Abcam ab133997) and components of the extracellular matrix, such as MMPs and TIMPs (Abcam ab134004), were performed as per the manufacturer’s instructions and tested for the effects of our treatments on the regulation of levels of human inflammatory markers, cytokines, and components of the extracellular matrix.
5
Macrophage Secretome Modulates Chondrocyte MMP
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The CMs of the different macrophages (M0, M0 + iPSC-EV, M1, and M1 + iPSC-EV) were collected and centrifuged at 300×g for 5 min to remove cell debris. These collected CMs were then applied to the chondrocytes at 50% concentration (1:1 chondrocyte medium with 5% FBS: macrophage-CM). Chondrocytes were incubated in the media for 24 h. The control group was cultured in a 100% conventional chondrocyte medium. The medium was replaced with fresh serum-free medium and each group was cultured for an additional 24 h. The CMs of the chondrocytes (five groups) were harvested for MMP array analysis. A human MMP antibody array membrane (ab134004, Abcam) was used, following the manufacturer's instructions. Chondrocyte cell samples were harvested for total protein isolation and western blot analysis.
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