Quantamatrix Multiplexed Assay Platform (QMAP) analyses for the measurements of serum biomarkers were performed using shape-encoded magnetic microdisks and a QMAP 2.0 (Quantamatrix Inc., Seoul, Korea). Microdisks were hybridized with capture antibodies and reacted with samples and detection antibodies. Target-specific antibodies were purchased (R&D systems, Minneapolis, MN, USA) and product information (capture/detection antibody) is as follows: FGF21 (#DY2539/#BAF2539), GDF15 (#MAB957-100/#BAF940), adiponectin (#DY1065/#DY1065), leptin (#MAB398-100/#BAM398), retinol binding protein 4 (RBP4) (#MAB33781-100/#DY3378), and dickkopf-related protein 1 (DKK1) (#MAB10962-100/#BAF1096). After washing, microdisks were conjugated with streptavidin-R-phycoerythrin (Prozyme, Agilent Technologies, Santa Clara, CA, USA; #PJRS20). Fluorescence microscopic images were obtained using QMAP 2.0 and fluorescence intensities of all microdisks in the image were automatically analyzed using the software provided by QMAP.
Streptavidin r phycoerythrin
Manufactured by Agilent Technologies
Sourced in United States
Streptavidin-R-phycoerythrin is a fluorescent labeling reagent used in various bioanalytical techniques. It consists of the protein streptavidin conjugated to the fluorescent dye R-phycoerythrin. This conjugate enables the detection and visualization of biotinylated molecules.
Automatically generated - may contain errors
2 protocols using streptavidin r phycoerythrin
1
Multiplexed Serum Biomarker Quantification
2
Tetramerization and Phenotyping of HLA-B*5801-TW10
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HLA-B*5801-TW10 monomer was received from the National Institutes of Health Tetramer Core Facility. A 10-step tetramerization protocol of this monomer was performed as recommended by the manufacturer. Briefly, 0.32 µl Streptavidin-R-Phycoerythrin (Agilent Technologies) was added to each μg of TW10 monomer in 10 steps at 10-min intervals. HST cells were stained with tetramer-R-PE for 20 min at room temperature and costained with anti–PD-1 (CD279)–Alexa Fluor 488, clone EH12.2H7, anti–CD8-BV605 (both from BioLegend), and Live/Dead Aqua Fixable cell stain (Invitrogen). Cells were then fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences) following the manufacturer’s instructions and then stained intracellularly with anti–perforin-PE/Cy7, clone dG9 (BD Biosciences) and anti-granzyme B-BV421, clone GB11 (BioLegend). Samples were run on an Attune NxT flow cytometer (Thermo Fisher Scientific) and analyzed using FlowJo software (version 10). Phenotypes of tetramer+ cells were determined after gating on viable CD8+ cells.
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