We quantified protein concentrations of interleukin (IL)-8/CXCL8 and IL-6 in cell-free conditioned medium, supernatant from saliva, sputum, and BAL fluid samples utilizing in-house enzyme-linked immunosorbent assay (ELISA) methods, as previously described.18 (link) Briefly, commercially available antibody pairs MAB208/MAB 206 and MAF208/MAF 206 (R&D Systems, Inc., Minneapolis, MN, USA) were used to detect IL-8 (detection range: 12.5–6,400 pg/mL) and IL-6 (detection range: 3–375 pg/mL). An intra-assay variation <10% was accepted for the duplicate samples. We also quantified protein concentrations of MMP-9 in cell-free supernatant in conditioned medium, saliva, sputum, BAL fluid, and serum samples utilizing a commercial ELISA kit (DouSet ELISA MMP-9 Kit; R&D Systems, Inc.). The analyses of MMP-9 were performed according to the manufacturer’s instructions, and an intra-assay variation <10% was accepted for the duplicate samples.
Douset elisa mmp 9 kit
Manufactured by R&D Systems
Sourced in United Kingdom
The DouSet ELISA MMP-9 Kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of matrix metalloproteinase-9 (MMP-9) in biological samples, such as cell culture supernatants, serum, and plasma.
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3 protocols using douset elisa mmp 9 kit
1
Quantification of Inflammatory Cytokines and MMP-9
2
Quantifying Cytokine and Protease Levels
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Measurements of interleukin 8 (IL-8/CXCL8) in BM from unexposed/exposed model and AM from exposed model were performed using an in-house ELISA [15 (link)]. The detection limit of the IL-8 assay was 50 pg/ml. Levels of matrix metallopeptidase 9 (MMP-9) and club cell protein in BM from unexposed/exposed models and AM from exposed models were measured using a purchased DouSet ELISA MMP-9 Kit and uteroglobin kit (R&D SYSTEMS®, UK), respectively. The detection limits of the MMP-9 and club cell protein assay were 31.2 pg/ml.
3
Measurement of Inflammatory Markers in Biological Fluids
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Measurement of IL-6 and IL-8 in saliva, sputum and BAL-fluid was performed using an in- house ELISA [15 (link)]. The lower detection limit of the IL-6 assay was 3 pg/ml in all compartments. The limit of IL-8 assay of saliva and sputum was 50 pg/ml and for BAL was 12.5 pg/ml.
MMP-9 and TIMP-1 in supernatant from saliva, sputum, BAL-fluid and serum were measured using purchased DouSet ELISA MMP-9 Kit and DouSet ELISA TIMP-1 Kit (R&D SYSTEMS®). The measurements of TNF-α in supernatant from saliva, sputum and serum were performed by purchased HS quantikine ELISA Kit (R&D SYSTEMS®). The analyses of MMP-9, TIMP-1 and TNF-α were performed according to the manufacturer. For all the duplicated samples, an intra-assay variation <10% (for TNF-α, <20%) was accepted.
MMP-9 and TIMP-1 in supernatant from saliva, sputum, BAL-fluid and serum were measured using purchased DouSet ELISA MMP-9 Kit and DouSet ELISA TIMP-1 Kit (R&D SYSTEMS®). The measurements of TNF-α in supernatant from saliva, sputum and serum were performed by purchased HS quantikine ELISA Kit (R&D SYSTEMS®). The analyses of MMP-9, TIMP-1 and TNF-α were performed according to the manufacturer. For all the duplicated samples, an intra-assay variation <10% (for TNF-α, <20%) was accepted.
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