Isolation and Culture of Murine and Human T Cells

The suspensions of spleen cells obtained from mice were prepared as reported by da Silva et al. [24 (link)]. The obtained cell suspensions were used to isolate CD4⁺ and CD8⁺ T cells using the Isolation Kit II from Miltenyi Biotec (Auburn, CA, USA), according to the manufacturer’s instructions. The negatively selected cells were stained with anti-CD3 phycoerythrin (PE) and anti-CD4 fluorescein isothiocyanate (FITC) or anti-CD8 FITC antibodies (BD Biosciences, San Jose, CA, USA) and analyzed by flow cytometry (Guava^® easyCyte, Millipore, Billerica, MA, USA). Purity grades of 94–96% were achieved.
The Jurkat E6.1 human acute leukemia T cell line was routinely grown in advanced Roswell Park Memorial Institute 1640 medium (Gibco^®, Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 2500 mg/L glucose, 10 mM HEPES, and antibiotics in a humidified 5% CO₂ atmosphere at 37 °C. Cell pellets were obtained by centrifugation at 300× g for 7 min at 4 °C. The cell concentration was maintained as recommended by the ATCC cell biology collection, a protocol that was successful adopted by several authors [50 (link),77 (link),80 (link),82 (link)].