Scanned images of the cytokine assays were read into the FIJI/ImageJ software [22 (link)]. The semi-automatic grid finding was based on pre-processing via Gaussian blur (size 4) and local maxima finding as described in Steinfath et al. [23 (link)]. Local maxima were detected via the FIJI function “Find Maxima” and exported to a file in the csv format. The csv file containing the local maxima was imported into the R programming environment to detect the corners and interpolate the grid with the pre-defined size between the corners. The interpolation routine took into account alleyways between blocks within the grid and adjusted grid positions to local maxima, where existent. For positions without detected local maxima, the interpolated values were used. Grid positions were read into the FIJI Microarray Profile plugin by Bob Dougherty and Wayne Rasband (https://www.optinav.info/MicroArray_Profile.htm , accessed on 21 December 2022), which was employed to quantify the integrated densities of the spots. Grid positions were annotated with the cytokine identifiers provided in the manuals of the manufacturer (Proteome Profiler Array from R & D Systems, Human XL Cytokine Array Kit, Catalog Number ARY022B and Human Kidney Biomarker Array Kit, Catalog Number ARY019).
Human kidney biomarker array kit
Manufactured by R&D Systems
Sourced in United States
The Human Kidney Biomarker Array Kit is a multiplex immunoassay designed to detect and quantify multiple biomarkers associated with kidney function and disease. The kit utilizes bead-based technology to simultaneously measure the levels of various proteins in a single sample. This product provides a comprehensive assessment of kidney health markers.
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5 protocols using human kidney biomarker array kit
1
Quantitative Cytokine Profiling via ImageJ
2
Quantitative Analysis of Kidney Cytokines in Organoid Cultures
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Untreated organoids and organoids treated with PAN, which had been subjected to analysis with the kidney cytokine assay Human Kidney Biomarker Array Kit (R&D Systems) and scanned, were image-analysed with ImageJ [27 (link)]. The Microarray Profile plugin by Bob Dougherty and Wayne Rasband (https://www.optinav.info/MicroArray_Profile.htm ) was employed to localize and quantify all spots on the array. As read-out, the integrated density generated by the Microarray profile plugin function Measure RT was used. In R/Bioconductor [28 (link)], the data resulting from the quantification was normalized with the Robust Spline Normalization from the Bioconductor lumi-package [29 (link)].
3
Cytokine Profiling of Kidney Organoids
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Cell culture supernatants of untreated kidney organoids and kidney organoids treated with PAN (2 days after treatment) were kept for cytokine array. Relative expression levels of specific human kidney-associated and urinary proteins were determined using the Human Kidney Biomarker Array Kit from R&D Systems. The cytokine assay was implemented as recommended by the manufacturer. In brief, membranes were blocked for 1 h on a rocking platform. Prepared samples were incubated in the Detection Antibody Cocktail for 1 h at room temperature. Thereafter, the antibody–sample mixtures were pipetted onto the membranes and were incubated overnight at 2–8 °C on a rocking platform. The membranes were washed thoroughly and Streptavidin-HRP was added onto the membranes, which were incubated for 30 min at room temperature. ECL detection reagent was used to detect the spots on the membrane.
4
Comprehensive Kidney Biomarker and Cytokine Profiling
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Human Kidney Biomarker Array Kit and Human XL Cytokine Array Kit (R&D Systems, Minneapolis, MN, USA) were used to assess the relative concentrations of 38 predefined markers of kidney damage and 105 inflammatory cytokines (total of 124 proteins, 19 were duplicated as they were evaluated in both kits). The full list of the proteins assessed can be found in supplementary Table S1 . Both kits were performed according to the manufacturer’s instructions. Briefly, each array kit consists of 4 nitrocellulose membranes with spotted capture and controls antibodies embedded in duplicates, each membrane allowing for 1 urine sample to be assessed (therefore 4 patients per kit). Three sets of each kit (12 membranes) were available, which allowed for the analysis of 12 different patients’ urine samples. The assays were conducted by performing 4 membranes at a time per day and subjects were intentionally divided to include at least one of each group in order to reduce selection bias. Due to the semi-quantitative nature of the membranes, we did not assess for intra/inter-variability, but we calculated Pearson’s correlation coefficient on the fold changes of the duplicated proteins.
For clarity, the Human Kidney Biomarker Array Kit will be further referred to as “Kit K” and the Human XL Cytokine Array Kit as “Kit C” throughout the study.
For clarity, the Human Kidney Biomarker Array Kit will be further referred to as “Kit K” and the Human XL Cytokine Array Kit as “Kit C” throughout the study.
5
Kidney Biomarker Profiling in PUUV Infection
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Proteome profiles of urinary samples derived from PUUV-infected and uninfected healthy individuals or cell-free supernatants derived from uninfected and PUUV-infected HRMCs (4 dpi) were analyzed by a Human Kidney Biomarker Array Kit (R&D Systems) according to manufacturer’s instructions. In brief, urine or cell culture supernatant samples together with biotinylated detection antibodies were added to membranes spotted with antibodies against 38 different kidney protein markers in duplicates. Detection of spot signal intensities, corresponding to the amount of bound proteins, was performed by using near infrared fluorescent IRDye800CW streptavidin and by scanning with an Odyssey CLx infrared imaging system (Li-Cor).
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