Plasma levels of MMP-9 were determined by enzyme linked immunosorbent assay (ELISA) using a human MMP-9 ELISA kit ( no. ab246539, Abcam, UK) according to the manufacturer’s instructions.
Human mmp9 elisa kit
Manufactured by Abcam
Sourced in United Kingdom, United States
The Human MMP9 ELISA Kit is a quantitative sandwich enzyme-linked immunosorbent assay (ELISA) designed for the measurement of human matrix metalloproteinase 9 (MMP9) levels in biological samples.
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5 protocols using human mmp9 elisa kit
1
Quantitative MMP-9 ELISA Assay
2
Comprehensive Oncology Protein Profiling
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Eighty-four different cancer-related proteins and the 43 kinases phosphorylation were measured with Proteome Profiler Human XL Oncology Array and Proteome Profiler Human Phospho-Kinase Array Kit (R&D Systems). Membranes were developed with Luminata Forte western HRP Substrate (Millipore) and images acquired with a Fujifilm LAS 3000 Imager. Dot intensity was quantified with ImageJ. Values were normalized to internal reference control. A restricted number of candidates were selected by plotting the DLK-A/DLK-C ratio of variation vs. internal reference control.
p53 activity was measured on nuclear extracts with p53 Transcription Factor Assay Kit (Abcam ab207225) following manufacturer’s instruction; Akt phosphorylation levels were measured with AKT1 + AKT2 + AKT3(pT308) ELISA Kit (ab176636) on total cellular extractions; Glucose and Lactate levels, VEGF and MMP9 secretion were measured on supernatant media with Glucose Assay Kit ab102517 (Abcam), L-Lactate Assay Kit (Abcam ab65331), Human VEGF-A ELISA Kit RAB0507 (Sigma) and Human MMP9 ELISA Kit ab100610 (Abcam). Each sample was read in duplicate. For p53, P-Akt, VEGF, MMP9 n = 3 for U3082MG, U3084MG, U3065MG. U3084S, n = 4 (p53, P-Akt), n = 3 (VEGF, MMP9, lactate, glucose).
p53 activity was measured on nuclear extracts with p53 Transcription Factor Assay Kit (Abcam ab207225) following manufacturer’s instruction; Akt phosphorylation levels were measured with AKT1 + AKT2 + AKT3(pT308) ELISA Kit (ab176636) on total cellular extractions; Glucose and Lactate levels, VEGF and MMP9 secretion were measured on supernatant media with Glucose Assay Kit ab102517 (Abcam), L-Lactate Assay Kit (Abcam ab65331), Human VEGF-A ELISA Kit RAB0507 (Sigma) and Human MMP9 ELISA Kit ab100610 (Abcam). Each sample was read in duplicate. For p53, P-Akt, VEGF, MMP9 n = 3 for U3082MG, U3084MG, U3065MG. U3084S, n = 4 (p53, P-Akt), n = 3 (VEGF, MMP9, lactate, glucose).
3
Quantifying E-Cadherin and MMP-9 in Cell Culture
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Human E-Cadherin ELISA Kit (cat. no. ab233611, Abcam), and Human MMP9 ELISA kit (cat. no. ab100610, Abcam) were used to detect E-cadherin and MMP-9 in the culture supernatants respectively. Samples were processed according to the manufacturer’s instructions. Untreated cells served as a control to determine the background levels of e-Cadherin and MMP under the experimental conditions used.
4
MMP9 Western Blot and ELISA
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MMP9 Western blot was carried out as previously described.32 (link) MMP9 secretion from Transwell cell culture supernatants (at day 4) was examined using the human MMP9 ELISA Kit (Abcam [ab246539]). Detailed information can be found in Supplementary Methods S1.3 .
5
Biomarkers for Post-Transplant Lung Dysfunction
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In order to validate the PEA results, CRH and MMP-9 in plasma were measured by ELISA kits according to manufacturer’s instructions: (CRH ELISA kit (OKEH00623), Aviva Systems Biology, San Diego, CA, US, Human MMP9 ELISA Kit (ab246539), Abcam, Cambridge, UK). The kits rely on standard sandwich enzyme-linked immunosorbent assay technology using specific antibodies. The optical densities of results were read at 450 nm. Sensitivity of the CRH and MMP9 assays were 4.9 pg/mL and 10 pg/mL respectively.
Plasma samples were taken at baseline following DLTx and of those 46 patients, 32 were analyzed again after 1 year. 6 patients were excluded due to re-transplantation secondary to BOS, another 5 died, and 3 were lost in follow up.
Microarray data from transbronchial biopsies was obtained from a study of 457 biopsies collected from consenting patients across 10 centers from the GEO dataset GSE150156.
From this set, gene expression microarrays were conducted according to previously described methods19 (link). Histologic analysis was undergone at the respective participating center according to the local standards of care, which allowed for categorization of the patients as either having chronic lung allograft dysfunction (CLAD) or non-CLAD .
Plasma samples were taken at baseline following DLTx and of those 46 patients, 32 were analyzed again after 1 year. 6 patients were excluded due to re-transplantation secondary to BOS, another 5 died, and 3 were lost in follow up.
Microarray data from transbronchial biopsies was obtained from a study of 457 biopsies collected from consenting patients across 10 centers from the GEO dataset GSE150156.
From this set, gene expression microarrays were conducted according to previously described methods19 (link). Histologic analysis was undergone at the respective participating center according to the local standards of care, which allowed for categorization of the patients as either having chronic lung allograft dysfunction (CLAD) or non-CLAD .
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