Cd8 t cell negative selection kit

PBMCs from healthy donors samples were isolated using Ficoll-Hypaque gradient centrifugation. T cells were obtained using SepMate™ PBMC Isolation Tube (STEMCELL, Vancouver, Canada). Isolated T lymphocytes were maintained in RPMI 1640 or ImmunoCult™-XF T cell Expansion Media (STEMCELL, Vancouver, Canada) with 50 U/ml interleukin-2 (PeproTech, Cranbury, NJ, USA). CD3⁺ T cells, CD4⁺ T cells, and CD8⁺ T cells were isolated using Pan T negative selection kit (Miltenyi Biotec, North Rhine-Westphalia, Germany), CD4⁺ T cell negative selection kit (Miltenyi Biotec, North Rhine-Westphalia, Germany) and CD8⁺ T-cell negative selection kit (Miltenyi Biotec, North Rhine-Westphalia, Germany), respectively, following the manufacturer’s instructions. Isolated T cells were activated by adding a CD3/28 T-cell activator (STEMCELL, Vancouver, Canada) and 50 U/ml interleukin-2. T cells were resuspended in CryoStor CS10(STEMCELL, Vancouver, Canada) at -80°C and thawed quickly in a 37°C water bath. All cells were grown in a humidified incubator at 37°C supplied with 5% CO₂ and tested regularly for Mycoplasma contamination.

Dendritic cells were inoculated at a MOI of 1 with the different viruses and collected 6 h later and cocultured with 1 × 10⁵ T cells/well; the T cells were either gBT-I CD8⁺ T cells purified from spleens of gBT-I transgenic mice using a CD8⁺ T cell negative selection kit (MiltenyiBiotec), or OT-II CD4⁺ T cells purified from the spleens of OT-II transgenic mice using a similar CD4⁺ T cell negative selection kit. For DC-OT-II cocultures, 200 nM ovalbumin_323–339 peptide (pOVA_323–339, Genscript) was added to the wells. After 24 h (gBT-I) or 48 h (OT-II) of coculturing, T cell activation, and differentiation was determined by ELISA by measuring IL-2, IL-4, and IFN-γ in the supernatants (28 (link)), and by flow cytometry using antibodies against CD4, CD8, CD25, and CD69 (all from BioLegend).