Human upar quantikine elisa kit

We generated the PLAUR variants rs2302524 and rs4760 (Supplemental Figure 19) using the GeneArt site-directed mutagenesis system (Thermo Scientific) and WT PLAUR (NCBI’s RefSeqGene LRG_637 and RefSeq NG_032898.1) cloned into a pCMV6-entry vector (Origene).
Equal amounts (12 μg) of plasmid DNA encoding vector control, human reference, or the PLAUR missense variants were transfected into HEK293T cells (CRL-3216; ATCC) using the FuGENE 6 transfection reagent (E2691; Promega). The conditioned media and cells from each plate were harvested 48 hours after transfection for performing the following: (a) assess uPAR distribution with immunofluorescence staining of cells using monoclonal uPAR antibody to uPAR domain 2 (NBP2-62800, 1:400, Novusbio) and membrane marker P-cadherin (ab16505; 1:100, Abcam); (b) quantification of gene expression using real-time quantitative PCR testing; and (c) suPAR measurement in the supernatant using the Human uPAR Quantikine ELISA Kit (DUP00; R&D Systems).
We performed hydrodynamic tail-vein injection of plasmid DNA encoding reference human PLAUR (n = 5), PLAUR variant rs2302524 (n = 9), and PLAUR variant rs4760 (n = 7) in 8-week-old C57BL/6J female mice and measured serum suPAR levels 24 hours after injection using the Human uPAR Quantikine ELISA Kit.

For this study, the Human uPAR Quantikine® ELISA kit (R&D Systems, Minneapolis, USA) was used according to the manufacturer’s instructions. The measurement of the optical densities was performed by the use of the “Infinite 200 Pro” plate reader (Tecan Group Ltd., Männedorf, Switzerland). In this study, all suPAR measurements were performed in duplicate. The suPAR concentrations were determined by calculating the average optical density value of the two wells with the same sample and determining the suPAR value by the use of the interpolated standard curve.

Human serum levels of the suPAR were determined using a Human uPAR Quantikine ELISA Kit (DUP00, R&D, Minneapolis, MN) according to the manufacturer’s protocol. Briefly, a capture antibody was pre-coated onto microplate wells and incubated overnight at room temperature (RT). Standards and samples were introduced to the wells and incubated for 2 h at RT. After washing, a biotinylated detection antibody was added to each well and incubated for 2 h at RT, followed by the addition of 100 μL streptavidin horseradish peroxidase (HRP) and incubation for 20 min at RT. A chromogen TMB substrate solution was added to the wells and incubated for 30 min at RT. Fifty microliters of stop solution was added to each well and read at 450 nm within 30 min. The method for the mouse plasma suPAR was performed according to the Mouse uPAR DuoSet (DY531, R&D). Ten individual samples at indicated time points were randomly selected for testing.