Rosettesep human cd8 t cells enrichment cocktail

Fresh peripheral blood CD8⁺ T cells from healthy donors were enriched by negative selection using the RosetteSep Human CD8⁺ T Cells Enrichment Cocktail (StemCell Technologies) following manufacturer's protocol. Purified CD8⁺ T cells were stained with anti-CD8 APC and anti-HLA-DR PE antibodies (Immunotools) and sorted with a FACS Aria II^u flow cytometer (BD Biosciences), obtaining the following subsets: CD8⁺HLA-DR⁺ and CD8⁺HLA-DR⁻. Both subsets were collected into complete RPMI 1640 medium containing 50% FCS and washed before further experiments. The purity of each population, determined by flow cytometry, was always over 95%. For CD8⁺ cells depletion, peripheral blood mononuclear cells from healthy donors were isolated by density gradient, and CD8⁺ lymphocytes were depleted after staining with anti-CD8 APC (Immunotools). CD8⁺ depletion was ~99% effective.

Human peripheral blood samples were collected in lithium heparin coated vacutainers (BD Vacutainer, 367886) from patients and healthy donors. Three synovial fluid samples were also collected under sterile conditions by the clinicians. CD8⁺ T cells were then isolated from blood and synovial fluid samples using RosetteSep Human CD8⁺ T Cells enrichment cocktail (StemCell Technologies Inc, BC, Canada) followed by Ficoll-Hypaque density gradient (HiSepTM LSM 1077, HIMEDIA, India) according to manufacturer’s protocol. Isolated plasma was stored in −80 °C for future use. Purity of isolated CD8⁺ T cells from blood samples was evaluated by flow cytometry (BD LSRFortessa) using FITC-anti-CD3 and PE-anti-CD8a antibodies (TONBO biosciences, San Diego, CA) and FACS Diva or FlowJo V10 software (Supplementary Fig. 1a). For flow cytometric analysis minimum of 10,000 viable events (CD8⁺ T cells) were acquired per sample.