Aim 5 media

2.5 x10⁵ PBMCs in a total volume of 100 μL of AIM V media (Sigma-Aldrich) were placed per well in flat bottom plates (Thermo-Fisher). For stimulation, PBMCs were either supplemented with 20 μL of Kv or recombinant protein (20mg/ul of RPMI). Plates were incubated for 36 hrs at 37°C and 5% CO₂ and spun at 500 g for 1 min at 21°C. Supernatant was aliquoted for immediate analysis.

Peripheral blood mononuclear cells (PBMCs) were isolated within 24 h of blood collection using the Ficoll (Amersham Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation method. The isolated PBMCs were then suspended in a freeze medium containing 10% DMSO (Sigma-Aldrich, St. Louis, MI, USA) and 90% heat-inactivated foetal calf serum (GIBCO, Life Technologies, USA) or 90% human AB serum (Sera Laboratories International, Sussex, UK) and frozen at a speed of 1 °C/min to −80 °C and subsequently stored in liquid nitrogen. Upon thawing, each vial of the frozen PBMCs was thawed rapidly in a 37 °C water bath and re-suspended gently in AIM-V media (Life Technologies, Grand Island, NY, USA) supplemented with 5% normal human serum (HS, Sigma-Aldrich, St. Louis, MI, USA) (complete AIM-V media).