Macs cd8 t cell isolation

CD4⁺ T cells and CD8⁺ T cells were negatively sorted from the spleens and lymph nodes with the MACS CD4+ T Cell Isolation (130–090-860) and MACS CD8+ T Cell Isolation (130–104-075) Kits (Miltenyi Biotec, Bergisch Gladbach, Germany).
For in vitro proliferation, 5 × 10⁵ isolated CD4⁺ and/or CD8⁺ T cells in 200 μl proliferation medium (RPMI supplemented with 10% FCS, 2 mM L-glutamine and 50 units/ml penicillin/streptomycin) were added in duplicate to 96-well plates precoated with anti-CD3 antibody (clone 2C11, 5 μg/ml) and soluble anti-CD28 (clone 37.51, 1 μg/ml; BD Pharmingen) was added. For TCR-independent T cell stimulation, 10 ng/ml phorbol 12,13-dibutyrate (PDBu) and 125 ng/ml of the calcium ionophore ionomycin were added to the media. Cells were harvested on filters after a 48-h stimulation period, pulsed with H3-thymidine (1 mCi/well) in the final 16 h and the incorporation of H3-thymidine was measured with a Matrix 96 direct β counter system.
IL-2 and IFN-γ production in mouse T cells after antibody stimulation was determined by BioPlex technology (BioRad Laboratories) from the supernatant.

WT mice were transported from the animal facility to our laboratory and sacrificed immediately. Spleen and lymph nodes of WT mice were isolated, mashed through a 40 µm cell strainer (NeoLab, ST292712340) and incubated 5 min with ACK-lysing buffer (Life Technologies, A10492-01) at room temperature. The process was stopped with MACS buffer (1× PBS, 2% FBS and 2 mM EDTA) and after a centrifugation step the cells were counted and resuspended in MACS buffer for subsequent MACS CD8⁺ T cell isolation (Miltenyi Biotech, 130-104-075) according the manufacturer’s instructions. The whole process from organ isolation to single cell suspension took approximately 1.5 h.
None of the cells were stored/frozen and reused later.
An aliquot of the cells was stained with trypan blue (Life Technologies, #15250-061) to discriminate between alive and dead cells and counted with a Neubauer improved chamber using a microscope (Leica, Wetzlar). The median cell yield per mouse (splenocytes and lymphnodes) was 1.2 × 10⁸ and the CD8⁺ T cell yield ~9 × 10⁶ per mouse. Cell viability was >95% and the purity of the CD8⁺ T cell fraction was >90% (detected with FACS). Before using the cells for the assay they were incubated with 1 µM CFSE (Biolegend, #423801) in PBS for 5 min at 37 °C. Afterwards the cells were pelleted and resupended in assay medium (see below).