Figure 4

Proseek analysis of TNF levels in plasma. (A) The performance of the Proseek TNF assay was evaluated by comparing series of dilution curves of TNF spiked in either Calibrator diluent buffer or mouse EDTA plasma. Recombinant TNF was spiked in Calibrator diluent buffer or mouse EDTA plasma and measured by Proseek TNF assay. The fold dilution is indicated on the x-axis and the signal is plotted as crossing point (Cp) values. The very similar curves illustrate full recovery in plasma, the linear range shows assay efficiency and the hook effect at 10× dilution indicates assay saturation. (B) TNF levels in plasma after pMCAO. Plasma samples were obtained 30 minutes after pMCAO and measured in the indicated dilutions (100×, 10× and undiluted). As a positive control to ensure assay performance TNF was spiked in control plasma (Calibrator diluent buffer, 10,000×, 1,000× and 100×) and as negative control plasma from a naïve control mouse (100×, 10× and undiluted). No change in TNF in plasma was observed 30 minutes after pMCAO. (C) TNF levels in plasma after pMCAO. Plasma samples were obtained 6, 12 or 24 hours after pMCAO and TNF levels were analyzed at the indicated dilutions (10,000×, 1,000×, 100× and 10×). The TNF level was at background levels and no change in TNF in plasma was observed at the indicated time points after ischemia. Plasma inhibition was introduced in the assay setup, demonstrated by lower signal in the high plasma concentration compared to the buffer level. In (A), (B) and (C) error bars indicate SD.