Detection of alanine transaminase (ALT) and aspartate transaminase (AST) in serum were performed by an fully automatic biochemical analyser (Siemens Advia 1650). Malondialdehyde (MDA) in liver homogenate was tested by the Enzyme linked immunosorbent assay (Becton, Dickinson) according to manufactor' s protocol.
Enzyme linked immunosorbent assay
Manufactured by BD
Sourced in United States
Enzyme-linked immunosorbent assay (ELISA) is a laboratory technique used to detect and quantify specific proteins or other molecules in a sample. It utilizes the high specificity of antibodies to bind to target analytes and the catalytic activity of enzymes to produce a measurable signal. ELISA is a widely used method in various fields, including diagnostics, research, and drug development.
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Lab products found in correlation
Advia 1650, by Siemens (1 mentions)
Cytometric bead array, by BD (1 mentions)
Hank s balanced salt solution, by Thermo Fisher Scientific (1 mentions)
Phorbol myristate acetate (pma), by Thermo Fisher Scientific (1 mentions)
Ionomycin, by Thermo Fisher Scientific (1 mentions)
Anti cd3, by Thermo Fisher Scientific (1 mentions)
Anti cd28, by BD (1 mentions)
Histopaque, by Merck Group (1 mentions)
Phorbol myristate acetate (pma), by Bio-Techne (1 mentions)
Luminex human assay, by Thermo Fisher Scientific (1 mentions)
Serum separator tube, by BD (1 mentions)
Mda assay kit, by Cayman Chemical (1 mentions)
Griess assay, by Cayman Chemical (1 mentions)
11 protocols using enzyme linked immunosorbent assay
1
Serum Enzyme and Oxidative Stress Assay
2
Neonatal Intensive Care Biomarker Analysis
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The biochemical and hematological findings of all study subjects were assessed by the results of routine blood tests on admission to the NICU. The residual serum samples were stored at À30 C until the following analysis. The concentrations of chemokines (CXCL8, CXCL9, CXCL10, CCL2 and CCL5) were determined by a cytometric bead array (Becton Dickinson, San Jose, CA, USA). The TGF-b1 level was determined by an enzyme-linked immunosorbent assay (Becton Dickinson, San Jose, CA, USA). The HA and KL-6 levels were measured by a latex agglutinationturbidimetric immunoassay (Fujirebio Inc., Tokyo, Japan) and chemiluminescent enzyme immunoassay (Eisai, Tokyo, Japan), respectively. 15, 16 The detection limits of each chemokine and TGF-b1 were 4 pg/ml and 100 pg/ml, respectively.
3
Bronchoalveolar Lavage Fluid Analysis
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BALF was obtained post-exsanguination of mouse. A cross-section incision was made on the exposed trachea. Following, a cannula was inserted, and the lung was flushed 3–4 times with 1 mL of Hanks’ Balanced Salt Solution (Gibco, Billings, MT, USA). Recovered fluid was placed into a 1.5 mL Eppendorf tube. BALF was subsequently centrifugated at 300g for 6 minutes and then at 5000g for 30 seconds. Fluid was separated from the pelleted lung cells. BALF IL-6 was quantified by enzyme-linked immunosorbent assay (BD Biosciences, Franklin, Lakes, NJ.) as per manufacturer instructions.
4
T Cell Activation and Culture Protocol
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Vero E6 (provided by Wendy Maury, PhD, University of Iowa), Vero (American Type Culture Collection), Ramos-B (American Type Culture Collection), and Jurkat E6.1 T cells were maintained in medium, as described elsewhere [16 ]. Human peripheral blood mononuclear cells (PBMCs) were obtained from healthy blood donors, as described elsewhere, or from the University of Iowa DeGowin Blood Center and were prepared using Histopaque (Sigma-Aldrich) purification [17 (link)]. CD3+ or CD4+ T cells were further purified (>90%) from PBMCs by negative selection (R&D Systems), with purity assessed by means of flow cytometry (Supplementary Figure 1 ). Jurkat cells were activated with 1-µg/mL anti-CD3 (Invitrogen) and anti-CD28 (BD Biosciences) or 50-ng/mL PMA (Tocris Bioscience) and 1-µg/mL ionomycin (Alfa Aesar). Primary cells (1 million cells/mL) were activated with 200-ng/mL anti-CD3 (Invitrogen) or 5-ng/mL PMA and 100-ng/mL ionomycin. Cell activation was assessed by measuring interleukin 2 release 16 hours after stimulation, using enzyme-linked immunosorbent assay (BD Biosciences) as described elsewhere [18 (link), 19 (link)].
5
PBMC Isolation and Cytokine Profiling
6
Priming Rat Bone Marrow Cells
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Undifferentiated rat bone marrow cells were primed with 4 ng/ml rat TNFα for 30 minutes, then stimulated with either 100 mg/ml MPO-ANCA or control IgG isolated from EAV or healthy rats, respectively. For inhibition experiments, cells were pretreated with R406 and/or vehicle for 30 minutes after TNFα-priming and prior to IgG stimulation. MCP-1 production was measured using a commercially available enzyme-linked immunosorbent assay (BD Biosciences, Oxford, UK), and reactive oxygen species production was measured by CellROX Deep Red assay (Invitrogen, Carlsbad, CA). Results were standardized to allow comparison between each of the biological replicate experiments.
7
Quantifying TNF Secretion in Cell Lines
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For measuring TNF secretion, L929 cells (2 × 104/well) or REH cells (1 × 106/well) were seeded in 96-well tissue culture plates and cultured overnight. The next day, cells were challenged with SM in the presence and absence of NaCl. The supernatant was collected and TNF was quantified using an enzyme-linked immunosorbent assay (BD Biosciences, Heidelberg, Germany). All groups were analyzed as triplicates (technical replicates).
8
Cell-free NKT Cell Activation Assay
9
Cytokine Profiling in Murine Peritoneal Fluid
10
Biochemical and Inflammatory Markers Evaluation
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Immediately after MVB withdrawal, blood samples were collected from the previously cannulated left carotid artery. Serum was obtained via centrifugation at 1500× g for 10 min. Alanine aminotransferase, aspartate aminotransferase (AST), creatinine, urea, triglycerides, total cholesterol, and HDL-C levels were measured using a semi-automated biochemical analyzer (Aker BIO-200). LDL-C and VLDL-C were calculated according to the Friedwald formula [5 (link)]. Serum NT, oxLDL, sVCAM-1, sICAM-1, IL-1β, and IL-6 levels were measured using enzyme-linked immunosorbent assays (BD Biosciences, San Jose, CA, USA). Malondialdehyde levels were measured using an MDA assay kit (Cayman Chemical, Ann Arbor, MI, USA).
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