Duoset ancillary reagent kit 2

Human MCP-1, sICAM-1, and TF were analyzed in EDTA containing samples using a Duoset ELISA (R&D System, Minneapolis, MN, USA) according to the manufacturer’s recommendations. Microplate-format colorimetric human MCP-1, sICAM-1, and TF solid phase ELISA immunoassays were established and validated. A DuoSet Ancillary Reagent Kit 2 (R&D Systems, Catalog # DY008) containing 96-well microplates, plate sealers, substrate solution, stop solution, plate coating buffer (PBS), wash buffer, and reagent diluent concentrate were used to establish the ELISA immunoassays.

The expression levels of CXCL1 released in the cell culture medium were detected by ELISA using the Human CXCL1 DuoSet ELISA (R&D System, cat no. DY275). For TNF-α was used Human TNF-α DuoSet ELISA (R&D System, cat no. D210), and IL-6 DuoSet ELISA (R&D System, cat no. DY206) for IL-6 quantification along with DuoSet Ancillary Reagent Kit 2 (R&D Systems, cat no. DY008).

A patient cohort of healthy controls and CKD patients with a measurement of serum calcification propensity determined by a one-half maximal transition time (T50) of in vitro transformation from primary to secondary calciprotein particles using a Nephelostar Plus nephelometer (BMG Labtech, Ortenberg, Germany) was previously described in detail [34 (link)]. The serum periostin levels were determined using a human Periostin DuoSet ELISA kit (#DY3548B, R&D Systems, Abingdon, UK) and a DuoSet Ancillary Reagent Kit 2 (R&D Systems, Abingdon, UK).

The human milk samples were thawed, and the nesfatin-1 level was analyzed immediately in the laboratory in Umeå, Sweden, using Enzyme-Linked Immunosorbent Assay (ELISA) kits, DouSet ELISA human Nesfatin-1/Nucleobindin-2, Cat no: DY5949, Lot: 325592 (R&D Systems Inc., Minneapolis, MN, USA) and DuoSet Ancillary Reagent kit 2, Cat no: DY008. The samples were measured in duplicate, and the average concentration was calculated from the standard curve in the ELISA software. The coefficient of variation (CV) (i.e., standard deviation (SD)/average) was calculated for the two duplicate values, and the samples were reanalyzed if CV was greater than 10%. The total concentration was the average value multiplied by the dilution. All samples were diluted 20 times, but a few samples were diluted up to 40 or 80 times before the concentrations were stable. Two control samples were included in all four plates to detect possible interplate variability. The average interplate CV for the two controls was 17.5%.

E. coli LPS-stimulated TNFα and IL-1β secretion from peritoneal macrophages isolated from WT or Spock1-Tg mice were measured using DuoSet Ancillary Reagent Kit2 (DY008; R&D Systems), mouse TNF-alpha DuoSet ELISA (DY410-05; R&D Systems) and mouse IL-1beta/IL-1F2 DuoSet ELISA (DY401-05; R&D Systems) according to the manufacturer’s instructions. Absorbance was measured at 450 nm using a microplate reader (Bio-Rad Laboratories, Hercules, California, USA). For quantification, peritoneal macrophages after stimulation were collected using 100 µL of CytoBuster Protein Extraction Buffer (Merck) for protein extraction. Protein concentration was measured using the Quick Start Bradford Protein Assay Kit (Bio-Rad Laboratories) and 4× LeammLi Sample Buffer (Bio-Rad Laboratories) according to the manufacturer’s protocol.