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).

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