U plex biomarker group 1 kit

Cytokines secreted by macrophages and MGC were measured using a custom mouse U-PLEX Biomarker Group 1 kit (Meso Scale Discovery, Rockville, MD), which is a multiplex sandwich immunoassay consisting of biotinylated capture antibodies and SULFO-TAG conjugated detection antibodies. The 96-well plate assay was completed according to manufacturer protocol. The plate was washed with 300 μl/well using a Thermo Wellwash 4 MK 2 and shaken at approximately 715 RPM on a Thermo 4625 shaker. Electrochemiluminescence was measured using the MESO QuickPlex SQ 120 with Discovery Workbench 4.0 software. Secreted protein concentrations were calculated using a four-parameter logistic standard curve. Some concentrations were below the limit of detection and could not be estimated from the curves, particularly in unstimulated cells. These nondetect values were considered to be zero for graphing and statistical analysis.