Under terminal anaesthesia, blood was collected directly from the right atrium into heparinised tubes, was centrifuged at 3000 rpm for 15 min at 4 °C and the remaining plasma aliquoted and stored at −20 °C. Samples were then analysed for CCL2 and CXCL1 using R&D systems sandwich-type duo set ELISA kits (DY479, DY453) while IL-1β was analysed using a Quantikine kit (R&D systems, Minneapolis, MN, USA, MLB00C). A standard protocol was followed as previously described [30 (link)] except for IL-1β, which was as per manufacturer’s instructions with minor modifications. To ensure that all cytokines were reliably quantifiable using the appropriate standard curves, samples were serially diluted for CCL2 and CXCL1 (1/9, 1/81 and 1/243). Blood and brain samples were also assayed for the presence of human IL-1RA using an R&D Systems quantikine assay (DRA00B), performed according to manufacturers’ instructions (standards 0–2000 pg/ml). Hippocampal/thalamic tissue punches were homogenised in 150 mM NaCl, 25 mM Tris-HCl and 1% Triton X100 at pH 7.4 before centrifugation at 14,000 rpm for 10 min. Supernatants were diluted 1 in 2 in assay diluent in wells pre-coated with anti-human IL-1RA polyclonal antibody.
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