Macs bead separation

Whole blood from Pre-Ex and Post-Ex timepoints only were used to isolate PBMCs using density gradient centrifugation. Blood was diluted 1:1 with Hanks Balance Salt Solution (HBSS), and then layered carefully on top of Histopaque 1077 (Sigma, Dorset), before centrifuging at 400g for 40 ​min at 21 ​°C. The PBMC layer was aspirated and then washed three times with HBSS, by centrifuging steps at 300g for 10 ​min. Approximately 3 million cells per time point were then used to enrich CD8⁺ T-cells by negative selection using MACS® bead separation (Miltenyi Biotec, Surrey, UK). PBMCs were incubated with a biotin-antibody cocktail (anti-CD4, CD15, CD16, CD19, CD34, CD36, CD56, CD123, TCRγ/δ, and CD235a), followed by a CD8⁺ T-cell microbead cocktail (anti-CD14, CD61 and anti-biotin) for 10 ​min at 4 ​°C, with intermittent washes using column wash buffer (PBS supplemented with 0.5% bovine serum albumin and 2 ​mM EDTA; pH ​= ​7.2). CD8⁺ cells from both time-points were eluted by negative selection using three separate MiniMACS LD-column passes, using an excess of de-gassed column buffer (Miltenyi Biotec, Surrey, UK). CD8⁺ T-cell enrichment was confirmed by flow cytometry staining analysing the CD3⁺CD8⁺ lymphocyte population (>95% purity).