Easysep magnetic separation kits

Peripheral blood mononuclear cells (PBMCs) were prepared from whole blood by Ficoll gradient centrifugation (Lymphoprep, STEMCELL Technologies) and used directly for total T cell enrichment by negative magnetic selection using Easysep magnetic separation kits (STEMCELL Technologies). Cell suspension was stained with anti-CD4 (RPA-T4), anti-CD8 (RPA-T8), anti-CD25 (clone 2A3), anti-CD45RO (UCHL1), anti-CD45RA (HI100) and anti-CD127 (hIL-7R-M21, all from BD Biosciences) for 30 minutes at 4°C. Naïve CD4⁺ T cells (CD4⁺/CD25^neg/CD127⁺/CD45RO^neg/CD45RA⁺) and naïve CD8⁺ T cells (CD8⁺⁺/CD25^neg/CD127⁺/CD45RO^neg/CD45RA⁺) were sorted on a FACSAria (BD Biosciences). Sorted cells were plated in 96-well round-bottom plates (Corning) and cultured in RPMI 1640 medium supplemented with 5 % Human serum, 2 nM L-glutamine, 5 mM HEPES, and 100 U/ml penicillin, 100 mg/ml streptomycin, 0.5 mM sodium pyruvate, 0.05 mM nonessential amino acids, and 5% human AB serum (Gemini Bio-Products). Cells were seeded (30,000-50,000/wells) into wells pre-coated with anti-human CD3 (2 mg/ml, clone UCHT1, BD Biosciences) along with soluble anti-human CD28 (1mg/ml, clone 28.2, BD Biosciences) in the presence or absence of human IFN-b (500 U/ml: Pestka Biomedical Laboratories) or IL-27 (100 ng/ml: BioLegend) without adding IL-2.