MSD V-PLEX Assay Protocol

The assay was performed in accordance with the manufacturer's instructions (MSD kit reference K15048D-1). Plasma samples were diluted 1:2 using Diluent 41. The standards, blank, and samples (cortical lysates or plasma) solution were measured in duplicates, with 50 μl of each solution added to an allocated well within the customized 96-well plate. The plate was sealed and incubated at room temperature on a shaker for 2 h. After incubation, the plate was washed three times with wash buffer, and 25 μl of the diluted detection antibody was added to each well, the plate resealed, and incubated at room temperature on a shaker for a further 2 h. After antibody incubation, the plate was washed three times, as previously described, and then 150 μl of 2X read buffer T was added to each well. The plate was then placed on the MSD instrument and read immediately. The reading of the V-Plex plate was performed using the MSD SECTOR Imager 2400, according to the manufacturer's manual (MSD, USA). The plate had an MSD barcode that allowed the SECTOR Imager to detect the type of plate being run. The data generated was automatically analyzed with a template using the Discovery Workbench version 4.0 software. The cytokines levels were expressed in pg/ml for plasma and pg/mg of protein detected in a 100 mg/ml tissue lysate, determined using the Bradford protein assay. One-way ANOVA with Tukey's post hoc analysis was performed using the SPSS software.