Cd8 rosettesep

Peripheral blood mononuclear cells (PBMCs) were obtained from healthy community subject buffy coats (from blood drawn within 24 hours), under consent, from commercial vendors. PBMC were isolated from buffy coats using Lymphocyte separation medium (Corning, Amsterdam, The Netherlands) density centrifugation and CD8⁺ T-cell depleted using CD8⁺ RosetteSep (StemCell Technologies Inc., London, United Kingdom).

Peripheral blood mononuclear cells (PBMCs) were isolated from healthy community donor buffy coats (from blood drawn within 24 h) with appropriate consent and obtained from the UK National Blood Transfusion Service (Addenbrooke's Hospital, Cambridge, UK). PBMCs were isolated from buffy coats by Lymphoprep (Axis-shield, Dundee, UK) density centrifugation and CD8⁺ T cells were depleted using CD8⁺ RosetteSep™ (StemCell Technologies Inc, London, UK). Human leukocyte antigen-D related (HLA-DR) haplotypes were determined using an HLA sequence specific primer- PCR based tissue-typing kit (Biotest, Solihull, UK). PBMCs were frozen and stored in liquid nitrogen until required.

PBMCs were isolated from 50 healthy donor buffy coats and CD8+ T cells were depleted using CD8+ RosetteSep (StemCell Technologies). Cells were plated at a density of 4–6 million cells per mL in AIM-V medium (Gibco). On days 5, 6, and 7, cells were gently resuspended in 3 × 100 μL aliquots and transferred to each well of a round bottomed 96 well plate. Cultures were pulsed with 0.75 μCi [³H]-Thymidine (Perkin Elmer) in 100 μL AIM-V culture medium, and incubated for a further 18 h, before harvesting onto filter mats (Perkin Elmer), using a TomTec Mach III cell harvester. Counts per minute (Cpm) for each well were determined by Meltilex (Perkin Elmer) scintillation counting on a 1450 Microbeta Wallac Trilux Liquid Scintillation Counter (Perkin Elmer) in paralux, with low background counting.