Biomarkers were measured on EDTA plasma or serum. Intestinal fatty acid binding protein (I-FABP) was measured using the DuoSet ELISA Kit; sCD14 and IL-6 were measured by ELISA using Quantikine kits; sCD163 and sTNFRII were measured using Luminex high-performance assays (all R&D systems, Minneapolis MN). Serum (1,3)-β-D-glucan was measured using the Fungitell® kit (Associates of Cape Cod, Inc., Falmouth, MA). Other inflammation biomarkers were measured using the Luminex platform (Bio-Plex Pro™ Human Inflammation Panel 1, Bio-Rad Laboratories, Inc., Hercules, CA).
Luminex high performance assays
Manufactured by R&D Systems
Luminex high-performance assays are multiplex immunoassay platforms that allow simultaneous detection and quantification of multiple analytes in a single sample. The core function of these assays is to provide a flexible, efficient, and high-throughput solution for measuring a variety of biomolecules, including proteins, cytokines, and other targets.
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2 protocols using luminex high performance assays
1
Biomarkers of Inflammation and Intestinal Damage
2
Secreted Cytokine Profiling of CB Cells
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To explore the secreted cytokine profile of the CB populations, Luminex High Performance Assays (R&D Systems) were performed. The supernatant of both freshly sorted UTCs and UTC-derived clones after 24 h of stimulation with phorbol myristate acetate (PMA; 1 ng/ml; 16561-29-8; Sigma-Aldrich) + ionomycin (0.5 µg/ml; 56092-82-1; Sigma-Aldrich) was assessed with the Luminex Performance Human XL Cytokine Magnetic Panel 44-plex Fixed Panel (LKTM014; Bio-Techne). Following this, a custom mixed multiplex was used to determine the IFN-γ, granzyme B, GM-CSF, MIP-1α, MIP-1β, and IL-2 concentrations in the supernatant of both freshly sorted CTCs and UTCs after 24 h of stimulation with PMA + ionomycin. All assays were performed conforming to the manufacturer’s protocol, measured with the Bio-Plex 200 system (Bio-Rad), and analyzed with the Bio-Plex Manager software version 6.2. Levels below or above the detection level were set as the lower or upper detection level, respectively.
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