The levels of murine serum TNF-α, IL-6 were measured using a sandwich ELISA Kit (Quantikine M; R & D systems, Minneapolis, MN55413 USA). The assay was performed as per the detailed instructions of the manufacturer.
Quantikine m
Manufactured by R&D Systems
Sourced in United States
The Quantikine M is a solid-phase enzyme-linked immunosorbent assay (ELISA) designed for the quantitative measurement of specific analytes in cell culture supernates, serum, and plasma. It provides a precise, reproducible, and sensitive method for detecting and quantifying target molecules.
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7 protocols using quantikine m
1
Murine Serum Cytokine Levels
2
Quantification of TNF-α and IL-6 by ELISA
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The levels of TNF-α, IL-6 were measured from extracellular medium using a sandwich ELISA Kit (Quantikine M; R & D systems, Minneapolis, MN, USA). The assay was performed as per the detailed instructions of the manufacturer33 (link) for 4 independent times.
3
Ischemia-Reperfusion Injury Biomarkers
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TNF-α (pg/mL) values were checked before ischemia, in the first hour of ischemia, and in the first and fourth hours of reperfusion; at the end of reperfusion period plasma and tissue MPA values, MDA values, and sICAM-1 levels were checked. TNF-α assays were performed according to the method described by Pizarro et al. [11 (link)] using the quantitative sandwich enzyme immunoassay technique (Quantikine M, R&D Systems, Minneapolis, MN, USA). sICAM-1 levels were measured by single stage photometric enzyme immunoassay method. Lipid peroxidation in the lung tissue was evaluated by measuring the level of MDA, which is the end product of lipid peroxidation. MDA was measured in plasma and the lung as an indicator of free radicals. Plasma and tissue (lung) MDA levels were determined with spectrophotometry using thiobarbituric acid-reactive substances [12 (link)]. MPO, an enzyme found primarily within neutrophils, is a sensitive marker for quantifying neutrophil content in tissue. MPO activity, a sensitive marker of PML infiltration in tissues, was calculated by hydrogen peroxide dependent oxidation of tetramethylbenzidine, a reaction catalyzed by MPO [13 (link)].
4
Cytokine Measurement in Biological Samples
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Cytokine levels in the BAL fluid or supernatants from cultured cells were measured as previously described (25 (link)). Briefly, measurements of IL-4, IL-5, IL-10, and IL-12 were performed by ELISA (BD PharMingen, San Diego, CA, USA) with 96-well plates (Immulon 2; Dynatech, Chantilly, VA, USA); IL-13 measurements were performed using an ELISA kit (QuantikineM; R&D Systems, Minneapolis, MN, USA) for BAL fluid and ELISA set (DuoSet; R&D Systems) for In vitro cell culture supernatants, all following the manufacturer's protocol. The limits of detection were 1.5 pg/ml for IL-13, 4 pg/ml for IL-4 and IL-5, 10 pg/ml for IL-10 and IFN-γ, and 62.5 pg/ml for IL-13 ELISA set.
5
Spleen Cell Cytokine Secretion Assay
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1 × 106 spleen cells/mL in a final volume of 0.2 mL from individual mice (five animals per group) were plated in 96-well plates and incubated for 72 h at 37 °C along with 50 μg/mL CSA. The release of cytokines in the supernatants was calculated by ELISA kits (Quantikine M; R&D Systems, Minneapolis, MN, USA; TGF-β, e-Bioscience), keeping in mind the detection limit of these assays. These were <2.0, <2.5, <5.1, <1.5, and <4.6 pg/mL for IFN-γ, IL-12p70, TNF-α, IL-10, and TGF-β, respectively. The data demonstrated as mean ± SD of all 5 animals of the group under consideration.
6
Leptin Secretion in Differentiated 3T3-L1 Cells
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3T3-L1 cells were harvested 8 days after the initiation of differentiation. Subsequently, the medium was changed to stimulant-containing DMEM. Differentiated cells were stimulated by 2.5, 5 and 10 µg/ml of turmeric extracts for 24 hours. Subsequently, cells were washed twice with cold phosphate buffered saline and collected by scraping with a cell scraper into 50 mM Tris-HCl (pH 7.5) containing 1 mM EDTA. The harvested cells were sonicated for 5 seconds. After centrifugation at 13,000 g for 5 minutes at 48℃, the supernatants were assayed for leptin [22 (link)]. The concentration of leptin was measured by leptin immunoassay kit (Quantikine® M, R&D System, Minneapolis, MN), according to the manufacturer's instructions.
7
Cytokine Profile Analysis in BMDM
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The level of various cytokines in the culture supernatants of BMDM were measured using a sandwich ELISA kit (Quantikine M, R&D Systems) as per the instructions of the manufacturer.
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