Jurkat cell line

The acute T cell leukemia Jurkat cell line was purchased from ATCC. Cells over-expressing the anti-apoptotic protein Bcl-xL (Jurkat-Bcl-x_L) and cells lacking the expression of the pro-apoptotic protein Bak (Jurkat-shBak) were generated in our laboratory using a lentiviral system, as previously described [22 (link)]. These cell lines are completely resistant to doxorubicin, a drug that kills cells through the mitochondrial apoptotic pathway [22 (link)]. Cell lines were routinely cultured in RPMI 1640 medium with GlutaMAX (Life Technologies, Paisley, UK) supplemented with 10% fetal calf serum (FCS), penicillin (1000 U/mL) and streptomycin (10 mg/mL) (PanBiotech, Aidenbach, Germany) at 37 °C and 5% CO₂ using standard procedures. Human PBMCs from healthy donors were obtained by Ficoll density centrifugation from Leukopacks provided by the “Banco de Sangre y Tejidos de Aragón”. T-cell blasts were generated from PBMCs from the same donors by stimulation with PHA at 10 µg/mL overnight, with posterior washing and culture for at least 7 days in the presence of 30 IU/mL of IL-2, with medium changes each 48 h, as described in [41 (link)]. The use of human samples was approved by the “Comité Etico de la Investigación de Aragón” (CEICA) and written informed consent was obtained from each donor.

Human peripheral blood mononuclear cells were obtained from blood following centrifugation in Lymphocyte Separation Solution (Wisent Inc., St-Bruno, Canada) as previously described (24 (link)). Blood donors were male and female subjects between 18 and 65 years of age who had indicated that they had no known health issues and who had fasted for 12 h prior to the blood draw. T-cells were then isolated by negative selection using the human T-cell enrichment kit from Stem Cell Technologies (Vancouver, Canada) following the manufacturer’s instructions. Primary T-cells and the Jurkat cell line (ATCC, Manassas, VA) were cultured in RPMI-1640 supplemented with 10% FBS, 100 U/ml penicillin, 10 µg/ml streptomycin, 10 mM HEPES, d-glucose (up to 25 mM), and 1 mM sodium pyruvate at 37°C in a 5% CO₂ atmosphere. Jurkat cells are a human leukemic T-cell lymphoblast cell line that is often used to study T-cell functions. T-cells were stimulated with anti-CD3/anti-CD28 Dynabeads (1 × 10⁶ beads/ml) (Invitrogen) according to the manufacturer’s instructions in the presence of 30 U/ml IL-2 (Sigma-Aldrich) for up to 72 h before all experiments. HepG2 cells (ATCC) were cultured in Eagle’s minimal essential medium supplemented with 10% FBS, 100 U/ml penicillin, and 10 µg/ml streptomycin at 37°C in a 5% CO₂ atmosphere.