Measurements of PGE2 by fluorescence resonance energy transfer assay and of IL-1β, IL-6, IL-8, RANTES, and IL-12 p40 cytokines by enzyme-linked immunosorbent assay

Cell culture supernatants from PBMCs, monocytes, and platelets were collected and assayed for PGE2 by fluorescence resonance energy transfer (FRET) assay using PGE2 Homogeneous Time-Resolved Fluorescence assay (HTRF) Kit (Cisbio Bioassays) and Novostar plate reader (BMG Labtech) as previously described (21). PGE2 concentrations were calculated using four-parameter logistic fit using Origin software application (OriginLab). The detectable PGE2 range was from 10 to 5000 pg/ml. IL-1β, IL-6, IL-8, RANTES, and IL-12 p40 cytokines were measured in the monocyte cell culture supernatants using human Quantikine enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems) and Synergy2 Multi-Mode plate reader (BioTek Instruments).

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