Meso Scale Discovery Assay

The standards, antibody detection solution, and read buffer were prepared in accordance with the manufacturer's instructions (MSD Kit # K15048D-1). All reagents were warmed to room temperature before preparation. The multi-analyte lyophilized calibrator, supplied by MSD, contains the highest concentration of all the cytokines and served as the standard stock for the assay. The calibrator was reconstituted in 1,000 μl of Diluent 41 (MSD, USA), mixed by vortexing, and left for 5 min before serial dilution. A series of standards were prepared by serial dilution of the calibrator solution by adding 100 μl of the calibrator to 300 μl of Diluent 41 and vortex-mixed, and the process was repeated five times to generate a total of seven standards. Diluent 41 alone was used as the blank. The kit provided 10 separate detection antibodies, 60 μl of each at 50X stock solution. All detection antibody solutions were combined together (600 μl) and added to 2,400 μl of Diluent 45 (MSD, USA) to achieve 1:50 dilution. Reader buffer T 4X stock solution (MSD, USA) was diluted to 1:2 with distilled water.

The linearity of dilution was first performed on 16 wells of the customized 96-well plate using two cerebral cortices supernatant samples. One sample was from the vehicle (no surgery) group, while the other sample was from KA-treated (surgery). These samples were predicted to have the least and the highest amount of inflammatory changes, respectively, due to the extent of insult to the brain. The cortical supernatants or plasma samples were serially diluted to 1:2, 1:4, 1:8, and 1:16 using Diluent 41 (MSD). The observed values were assessed relative to the standard curve for all 10 inflammatory cytokines. The results were calculated based on the standard curve, and the observed concentration was multiplied by the dilution factor. The criteria for acceptable dilutional linearity were for the corrected observed concentrations to vary no more than 80% to 120% of the theoretical concentration between each serial dilution for each analyte (38).

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.