Msd sector imager 2400 plate reader

Maternal and fetal livers were analyzed for multiple cytokines using the rat 9-plex pro-inflammatory panel assay kit (Meso Scale Diagnostics [MSD], Rockville, MD), as previously described [31 (link)]. Liver homogenates were assayed for cytokines (IFNγ, IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, and TNFα) and chemokines (CXCL1, CXC-motif ligand 1) using the MSD multiplex platform. The raw data were measured as electrochemiluminescence signals with the MSD Sector Imager 2400 plate reader (Meso Scale Diagnostics, Rockville, MD) and analyzed using the Discovery Workbench 3.0 software (MSD). The lower limits of detection for the analytes in this assay were ≤2 pg/ml, except for: IL-1β (7 pg/ml), IL-5 and IL-6 (14 pg/ml), and IL-10 (20 pg/ml). The R² value for each standard curve was between 0.99 and 1.0. Samples being compared were run on the same plate and the % coefficient of variation of control replicates run on the same plate was between 2.3 % (TNFα) and 5.7 % (IFNγ). Liver cytokine data were corrected for protein concentration (Bio-Rad protein assay, Bio-Rad, Hercules, CA) and expressed as pg/g.

Approximately 100 mg kidney (cortex and outer medulla, frozen) per mouse was homogenized in 200μl lysis buffer (Tris buffered saline containing 0.25%Triton X-100 containing protease and phosphatase inhibitor cocktail, pH 7.4) in a Dounce homogenizer; supernatants were collected after centrifugation. Homogenates were assayed for cytokines and chemokines using a multiplex platform (Meso Scale Discovery, MSD) and the MSD Sector Imager 2400 plate reader (Meso Scale Diagnostics, Rockville, MD, USA). The electrochemiluminescent signals generated (raw data) were analyzed using the Discovery Workbench 3.0 software (MSD). The values were adjusted for protein concentration using the BioRad protein assay (Bio-Rad, Hercules, CA, USA).