Antioxidant enzyme activity was measured as follows: liver tissue was homogenized and total protein in the supernatant was quantified in accordance with the Bradford method. The value obtained was set as the standard. Catalase (CAT), superoxide dismutase (SOD), and glutathione-S-transferase (GST) enzyme activities were measured using ELISA assays (BioVision Inc.), using 100 μL of each enzyme. Liver samples were harvested to quantify levels of glutathione (GSH; an antioxidant substance) and malondialdehyde (MDA; an intermediate in tissue lipid oxidation). Levels were quantified by measuring chemical conversion using colorimetry.
Elisa assays
Manufactured by Abcam
Sourced in United States
ELISA (Enzyme-Linked Immunosorbent Assay) is a well-established analytical technique used to detect and quantify specific molecules, such as proteins, hormones, or antibodies, in a sample. ELISA assays employ antibodies and color change to identify and measure the target analyte.
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7 protocols using elisa assays
1
Antioxidant Enzyme Assays in Liver
2
Metabolic Adaptations to Exhaustive Swimming
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Following exhaustive swimming, the ICR mice were anesthetized using isoflurane and their blood was sampled via heart clipping. Organs including the liver and muscle were dissected from the euthanized mice. Serum was isolated from blood to measure levels of LDH, lactate, and non-esterified fatty acids (NEFA) using enzyme-linked immunosorbent assay (ELISA) assays (BioVision Inc., Milpitas, CA, USA). The glycogen levels in muscle were measured in using chemical ELISA assays (BioVision Inc.). Expression of peroxisome proliferator-activated receptor-delta (PPAR-δ), carnitine palmitoyltransferase-1 (CPT1), β-hydroxyacyl coenzymes A dehydrogenase (HADH), and uncoupling protein-3 (UCP3) in muscle were measured after RNA extraction using real-time-polymerase chain reaction (RT-PCR).
3
Quantifying VEGF and ICAM-1 Levels
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Concentrations of soluble VEGF and intercellular adhesion molecule 1 (ICAM-1) were determined in cell culture supernatants by ELISA assays (Abcam, Cambridge, Great Britain). The assays were performed according to the manufacturer’s instructions. Absorbance at 450 nm was measured by a Triad LT (Dynex, Chantilly, VA, USA) microplate reader. For absolute quantitation, VEGF and ICAM-1 standards were used to obtain the standard curves in each assay.
4
Lentiviral Overexpression of EPO in BM-MSCs
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BM-MSCs were resuspended at a density of 5 × 104 cells/mL and seeded in 6-well culture plates with 1.5 mL cell growth medium. The next day, cells were infected with the lentivirus for overexpressing EPO at multiplicity of infection (MOI) 10 for 2 h. Cells were washed with PBS (Gibco) and cultured in cell growth medium. At 48 h after infection, 200 μL culture medium supernatant was collected for ELISA assays according to the manufacturer’s instructions (Abcam). EPO concentrations were calculated according to a standard curve.
5
Assessing Urinary NGAL Levels
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In each group, after the injection of iodixanol or saline, 3 urine samples (0.5–2.0 mL) were collected at 1 h and 1, 3, 5, and 10 days. The samples were centrifuged at 3,200 rpm (4°C) for 20 minutes and subsequently placed into a −80°F freezer for storage. The concentration of urinary NGAL was analyzed using ELISA assays from Abcam (Cambridge, MA, USA), following the standard protocols. In order to minimize any confounding effects of urine flow rate, concentration levels of NGAL were normalized to urine creatinine concentrations (analyzed in local clinical laboratory) [28 (link)].
6
Serum and Luminal Cytokine Profiling
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The serum levels of total IGF-1 and leptin were measured by ELISA assays (Abcam, Cambridge, UK) according to the manufacturer’s procedures. Likewise, the levels of corticosterone in serum were measured by radioimmunoassay using a gamma counter (ICN Isomedic 4/600) following the guidelines of the manufacturer (MP Biomedicals, Santa Ana, CA, USA). The presence of IL-7, IL-12, IL-13, IL-17A, IL-21, IL-22, IL-23, CCL5, CXCL9, CXCL10, TNFα, IFNγ and TGFβ in the luminal fluid of the duodenum and colon was analyzed using a multiplex immunoassay based on fluorescence-encoded beads according to the manufacturer’s instructions (BioLegend, San Diego, CA, USA). The acquisition was performed in a BD FACSCanto™ II flow cytometer (BD Biosciences, San Jose, CA, USA). Off-line analysis was performed with LEGENDplex™ (BioLegend, San Diego, CA, USA) data analysis software (version 8.0), and results were expressed as the mean of the reporter fluorescence intensity PE (MFI) as a function of concentration (pg/mL). Each assay was performed with two technical replicates.
7
Evaluating Resveratrol's Effects on Angiogenic Factors
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PS30 cells (5x10 4 cells) were incubated in RPMI supplemented with 0.5% FCS, and treated with RES at different concentrations (10, 25 and 50 µM) with or without TRPA1 inhibitors (HC-030031, 50 µM; A-967079, 1µM) or activator (AITC, 30 µM) for 24h, in 12-well plates at 80% of confluence. The supernatants of the cell cultures were then collected, centrifuged and processed for HGF and VEGF detections by ELISA Assays using an immunoassay kit (Abcam, Paris, France) according to the manufacturer's instructions.
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