Bio plex pro human inflammation panel assay

Participants provided a 10 ml fasted blood sample between 08:00 and 09:00, having performed no strenuous exercise for 48 h. Blood was collected in to an anticoagulant free vacutainer (BD Vacutainer Systems, Plymouth, UK) and serum was prepared and stored in 2 ml aliquots at −20 °C until subsequent analysis. Seventy-eight serum samples were randomly selected for cytokine analysis from a list of participant numbers with BMI and age as a guide for selection (the investigator was blinded to all other participant information). Consequently, 39 young and 39 older (n=48 BF%<40; n=31 BF%⩾40) participants were selected for analysis. Serum concentrations of eight inflammatory cytokines (pro-inflammatory: IL-1β, IL-6, TNF-α, G-CSF; anti-inflammatory: IL-10, TGF-β1, TGF-β2, TGF-β3) and four chemokines (IL-8, MCP-1, MIP-1α, MIP-1β) were measured using multiplex luminometry. A 3-plex panel was used to measure TGF-β1, β2 and β3 concentrations (R&D Systems Europe Ltd, Abingdon, UK) and a Bio-Plex Pro Human Inflammation Panel Assay (Bio-Rad laboratories Ltd., Hemel Hempstead, UK) was used to measure the remaining nine cytokines, following the manufacturer's instructions. Samples were analysed using a Bio-Plex 200 system (Bio-Rad laboratories Ltd., Hemel Hempstead, UK).

Thirty-three participants provided a 10-mL overnight fasting (12 h) blood sample, without having performed vigorous exercise for 48 h before sampling. Blood was centrifuged (IEC CL31R; Thermo Scientific) for 10 min at 2700 × g (4°C), and serum was extracted and stored at −20 °C until subsequent analysis. Blood samples were unavailable for 17 participants (sampling failure or consent withheld).
Multiplex luminometry was used to measure the serum concentrations of 9 inflammatory cytokines [proinflammatory: IL-1β, IL-6, TNF-α, granulocyte-colony–stimulating factor (G-CSF), IFN-γ; anti-inflammatory: IL-10 and TGF-β1, -β2 and -β3] and 5 chemokines [IL-8, monocyte chemoattractant protein (MCP) 1, macrophage inflammatory protein (MIP) 1α, MIP-1β, and regulated on activation, normal T-cell expressed and secreted (RANTES)]. A 3-plex panel was used to measure TGF-β1, TGF-β2, and TGF-β3 concentrations (R&D Systems Europe Ltd.), and a Bio-Plex Pro Human Inflammation Panel Assay (Bio-Rad Laboratories Ltd.) was used to measure the remaining 11 cytokines, following the manufacturers’ instructions. Samples were analyzed with the use of a Bio-Plex 200 system (Bio-Rad Laboratories Ltd.).