Meso quickplex sq 120 plate reader

Colon homogenates were directly prepared from frozen tissue samples in ice-cold lysis buffer (water with 500 nM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholic acid, 0.1% SDS, 50 mM Tris HCl and 1 tablet of protease inhibitor/mL of solution) by sonication (Branson 450 digital sonifier®, 30% amplitude, 10 s) (15 mg tissue/100 μL of lysis buffer). Samples were subsequently centrifuged at 20,000 × g for 10 min. The supernatant was retrieved and kept at −80 °C until further analysis, and the pellet was discarded. The concentrations of the following colonic proinflammatory cytokines and chemokines were determined with a V-PLEX Plus Mouse Cytokine 19-Plex ELISA kit (MesoScale) following the manufacturer's instructions using a MESO QuickPlex SQ 120 plate reader (MesoScale): IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, KC/GRO, IL-10, IL-12p70, Il-15, IL-17AF IL-27p28/IL-30, IL-33, IP-10, MCP-1, MIP-1α, MIP-2 and TNF-α. The concentrations were normalized to the protein content, quantified with a Pierce™ BCA Protein Assay Kit (Sigma-Aldrich, St. Louis, USA).

Myeloperoxidase (MPO) release from neutrophils (5 independent donors), cultured in the absence or presence of WPs, was assessed after 24 h using a U-PLEX MPO assay and a MESO QuickPlex SQ120 plate-reader (both Meso Scale Diagnostics, Rockville, MD, USA). To trigger neutrophil activation, parallel cultures in the presence of the chemotactic peptide N-formyl-Mel-Leu-Phe (FMLP) were also performed.
Peripheral blood mononuclear cells (PBMCs) (3 independent donors) were cultured overnight at a density of 1 × 10⁶ cells/mL in complete RPMI 1640 medium supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco). The following day, 0.1 or 1 mg/mL WPs and/or 10 µg/mL lipoteichoic acid (LTA; as a stimulus representative of Gram-positive bacteria) were added. After 24 h, PBMCs were harvested (400× g for 5 min), and the conditioned medium was collected and assessed for a range of pro- and anti-inflammatory cytokines using a U-PLEX multiplexed cytokine array and a MESO QuickPlex SQ120 plate-reader.