Whole blood cytokine release assay

Phytohaemagglutinin (PHA) or Staphylococcus aureus enterotoxin A (SEA) were added to 1 ml aliquots of fresh whole blood in microfuge tubes and incubated at 37 °C for 16 hrs (1–72 hrs for time course experiments). Following centrifugation at 3000 × g for 8 minutes, plasma was collected and stored at −20 °C. Isolated PBMCs were resuspended in the same volume of RPMI media as the original blood volume and incubated under the same conditions as whole blood. ELISA kits (Biolegend, San Diego, USA) were used to measure IFN-γ, IL-10, TNF, IL-6, IL-4 and IL-12(p70) according to the manufacturer’s instructions. Plates were analysed using a Multiskan FC Microplate Photometer (Thermo Scientific, Waltham, USA). Results below the minimum detectable limits with 1:2 dilutions of plasma were recorded as zero.