Sodium heparin vacutainer tubes

Fasted blood samples (10 h-fasting) were taken from volunteers in sodium heparin vacutainer tubes (Greiner Bio-One Ltd., Gloucestershire, UK). Blood was layered over an equal volume of lympholyte (Cedarlane Laboratories Limited, Tyne & Wear, UK) and centrifuged at 930 × g for 15 min at room temperature. Plasma was removed into a sterile tube and stored at −80°C until further use. Cells were harvested from the interface, washed once, resuspended in RPMI 1640 medium (Autogen Bioclear, Wiltshire, UK) containing 0.75 mM glutamine (Autogen Bioclear, Wiltshire, UK), and the above steps were then repeated to achieve a lower degree of erythrocyte contamination. The pellet was finally resuspended in the same medium.

Fasted blood samples were taken from six healthy volunteers aged 60–73 years, in sodium heparin vacutainer tubes (Greiner Bio-One Limited, Gloucestershire, United Kingdom). The study was conducted according to guidelines laid down in the Declaration of Helsinki, and all procedures involving human subjects were approved by the Ethics Committee of the University of Reading. The ethics approval number was UREC 14/05. Written informed consent forms were obtained from all subjects. Blood was layered over an equal volume of lympholyte (Cedarlane Laboratories Limited, Burlington, Ontario, Canada) and centrifuged at 930 xg for 15 min at room temperature. Peripheral blood mononuclear cells (PBMCs) were harvested from the interface, washed once with PBS, and then resuspended in Roswell Park Memorial Institute (RPMI) 1640 medium (containing glutamine, Autogen Bioclear Ltd., Wiltshire, UK). These steps were repeated to achieve low contamination of erythrocyte. The pellet was finally resuspended in RPMI 1640 medium and cell numbers counted using trypan blue and a cell counter (Coulter, Fullerton, CA, USA). Cells were adjusted to the required concentration.