Blood collection tube

For DNA, blood was collected in EDTA-treated blood collection tubes (Becton Dickinson; BD, Oxford, UK). This was centrifuged to separate the plasma and cellular sections and then DNA was then extracted from the cellular section using a phenol chloroform extraction method. Both the plasma and DNA were stored at −80 °C. For the previously recruited subjects, DNA had been collected previously [30 ].
For PBMC extraction, blood (≤80 ml) was collected in sodium heparin treated blood collection tubes (BD) then layered onto Ficoll-paque Plus (GE Lifesciences, Buckingham, UK) in a 1:1 ratio and centrifuged (23 °C, 0.5 h, 480g). The PBMC layer was transferred into growth media (RPMI 1640 with l-glutamine and NaHCO3, with 10 % heat inactivated FBS, 100 units/ml penicillin, and 0.1 mg/ml of streptomycin [Sigma-Aldrich Company Ltd, Dorset, UK]) and centrifuged to pellet the PBMCs (24 °C, 10 min, 390g). These PBMCs were frozen in freezing media [Heat inactivated FBS (Life Technologies Ltd, Paisley, UK) with 5 % DMSO Hybri-max (Sigma-Aldrich)] at a concentration of ~1 × 10⁷ cells/ml. Frozen PBMCs were transferred to liquid nitrogen for long term storage. Recovery and viability after freezing were assessed by trypan blue staining and found to be high (normally >90 %).

Subjects were asked to abstain from vigorous physical activity, high-fat foods, and anti-inflammatory medications and supplements the day prior to their visit to the Paykel Clinical Trial Unit at the Liggins Institute. Subjects arrived fasted on two separate occasions a minimum of 14 days apart. Anthropometric data were collected before a catheter was inserted into an antecubital vein and a baseline sample (time 0) was taken followed by consumption of the test breakfast. Blood samples were collected hourly for 5 h post-meal in blood collection tubes (Becton Dickinson, Franklin Lakes, NJ, USA) for serum and ethylenediaminetetraacetic acid (EDTA) plasma, and processed as described previously [43 (link)]. Plasma supernatants were processed under laminar flow using pyrogen-free consumables. Plasma for chylomicron separation was kept at 4 °C and processed within 6 h and the remaining plasma was collected in pyrogen-free microtubes and stored at −20 °C.