Serum Cytokine Measurement Protocol

As we described previously (23 (link)), Arterial blood was placed in BD Vacutainer tubes (Becton Dickson, Franklin Lakes, NJ) after collection, hand-mixed, and stored at room temperature for 45 min, after which it was centrifuged (2,000 g) for 20 min at 4°C. The serum was removed with a micropipetter and stored at −80°C for later analysis. Interleukin (IL)-6 (RAB0311, Sigma, St. Louis, MO), Interleukin (IL)-1β (RAB0277, Sigma), and tumor necrosis factor (TNF)-α (ab100785, Abcam, Cambridge, UK) were measured in accordance with the manufacturer's instructions. For all assays, standards and unknowns were both measured in duplicate.