Heparin vacutainer tubes

For this nutrigenomic study, blood samples were collected from 12 volunteers randomly selected at the end of each 6-month dietary intervention. Blood was drawn in the morning after an overnight fast using heparin vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ, United States) and immediately centrifuged for 20 min at 1,500g at room temperature. The cell layer was collected and washed twice with sterile PBS, with centrifugation for 10 min at 300 g between each washing step. The obtained pellet of PBMCs was immediately frozen and kept at −80°C until used.

All clinical blood samples were handled under BS level 2 containment. For method optimisation experiments superfluous material from commercial bovine blood samples sent to PBD Biotech Ltd. for Actiphage testing was used. The clinical cervid blood samples originated from farmed deer in the East Midlands area of the UK. The deer were kept on outdoor pastures and in four different production groups. Blood was collected for diagnostic purposes by veterinarians in Heparin Vacutainer tubes (Beckton Dickinson, UK) and stored and transported at ambient temperatures (15–20°C) to ensure that the mycobacteria remained in an active growth phase required for productive D29 infection (28 (link)). The blood separation procedures were carried out within 12 h of collection. Samples for MAP blood ELISA assays were sent to Axiom Veterinary Laboratories (Devon, UK).

All peripheral blood samples were collected into heparin vacutainer tubes (Becton Dickinson). Tubes were spun (10 min, 3000 rpm, room temperature (RT)), and plasma was collected and stored at –80 °C. PBMCs were isolated by density gradient centrifugation using lymphocyte separation medium (TBDscience), washed twice with Ca/Mg-free PBS (BasalMedia) and cryopreserved in medium containing 90% fetal calf serum (FCS) (Gibco), 10% dimethyl sulfoxide (Sigma-Aldrich) until using.
PBMCs from 16 healthy controls, 9 asymptomatic subjects, 7 presymptomatic subjects, 8 mild COVID-19 patients and 8 moderate COVID-19 patients were processed for CyTOF. Plasma from 16 healthy controls, 9 asymptomatic subjects, 8 presymptomatic subjects, 9 mild COVID-19 patients and 10 moderate COVID-19 patients were processed for Olink. Total RNA extracted from PBMCs of 15 healthy controls, 9 asymptomatic subjects, 7 presymptomatic subjects, 7 mild COVID-19 patients and 9 moderate COVID-19 patients were used for RNA-seq. 28 SARS-CoV-2^pos subjects could be simultaneously analyzed by CyTOF, Olink and RNA-seq (Supplementary information, Table S1).