Cd8 positive selection kits

Human peripheral blood mononuclear cells (PBMCs) were isolated, as previously described Xystrakis et al. (31 (link)). CD4+ and CD8+ T cells were isolated using Dynabeads CD4 or CD8 positive selection kits (Invitrogen, Paisley, UK). Cells (1 × 10⁶/ml) were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine, and 50 μg/ml gentamicin, and stimulated with plate-bound anti-CD3 (1 μg/ml; OKT3) plus 50 IU/ml recombinant human IL-2 (Eurocetus, UK) in the presence or absence of 1,25(OH)₂D3 (BIOMOL research labs, UK), TGF-β1 (R&D Systems, UK), TGF-β2 (R&D Systems) or anti-TGF-β (R&D Systems) at indicated concentrations for 7 days. NIST SRM1648a Urban Particulate Matter (National Institute of Standards & Technology, USA) is an urban total particulate matter reference material with mean particle diameter 5.85 μm, collected in the USA. NIST was prepared as previously described by Pfeffer et al. (32 (link)).

PBMCs were isolated with the Ficoll-Paque density gradient centrifugation protocol. CD4⁺ and CD8⁺ T cells were isolated from PBMCs with Dynabeads CD4⁺- and CD8⁺-positive selection kits (Invitrogen), respectively. For isolation of EM, EMRA, CM, and SCM memory subpopulations, we stained PBMCs with the following antibody mix: anti-CD3-FITC (UCHT1, eBioscience), anti-CD45RA-eFluor450 (HI100, eBioscience), anti-CCR7-APC (3D12, eBioscience), and anti-CD95-PE (DX2, eBioscience). Cell sorting was performed on FACS Aria III, and all four isolated subpopulations were lysed with Trizol reagent immediately after sorting.