Quantikine elisa immunoassay kit

Laboratory parameters were assessed using the standard methods in our laboratory, including plasma NTproBNP (N-Terminal Pro-B-Type Natriuretic Peptide) (method: immunoenzymatic, Siemens, Marburg, Germany) and troponin (TNI) (method: immunoenzymatic, one-dimensional RxLMax, Siemens). The serum sTfR (mg/L) was measured from plasma frozen at −70 °C using immunonephelometry (Siemens Healthcare Diagnostics, Inc., Deerfield, IL, USA).
The Quantikine ELISA Immunoassay kit (R&D Systems, Inc., Minneapolis, MN, USA) was used to determine the levels of the remaining markers of interest. This assay employs the quantitative sandwich enzyme immunoassay technique. These were the following proteins: GDF-15 (also known as macrophage inhibitory cytokine-1 (MIC-1) (n = 79), CXL4/PF4 (Platelet Factor 4), follistatin (FS), MMP-9 (matrix metalloproteinases—gelatinase) (n = 159), lipocalin-2 (NGAL) (n = 159), myostatin (GDF-8), E-Selectin (CD62E), ICAM-1 (CD54 allele-specific), Il-6 and Il-22 (n = 159). The Synergy/HTX multi-mode reader analyzer was used for the measurement of absorbance. This research was carried out in the laboratory of the Department of Clinical Pharmacology at Wroclaw Medical University.

For the quantitative murine IL-6 assay, the Quantikine ELISA immunoassay kit (R & D System, cat# M6000B) was used. In brief, following treatment of the 23-day differentiated adipocytes in serum-free, the media was collected and used for the assay according to the manufacturer’s protocol.

The reduced glutathione levels were evaluated with the method illustrated by Siddiqui et al. (2021), while activities of the catalase (CAT), and glutathione-S-transferase (GST) were assayed using the methods of Toledo et al. (2011) (link). On the other hand, nitric oxide (NO) and the peroxidation marker of the lipids (MDA, malondialdehyde) were quantified following the referenced methods of Miranda et al. (2001) (link) and Fu et al. (2018) (link), respectively.
Moreover, the tumor necrosis factor-alpha (TNF-α) was estimated by Quantikine ELISA Immunoassay kit (R&D systems) as per the guidelines provided by the manufacturer. The main antibody was used to sensitize 96-well microplates for 30 min at room temperature. The tissue sample was then added, cultured for a further 30 min, and washed. After washing, a peroxidase-conjugated secondary antibody was applied and allowed to incubate. Then, an ELISA plate reader was used to calculate the cytokine concentration.