Ex neg lv225

JURKAT cell lines were generated from FADD-deficient JURKAT clone I2.1 (ATCC, CRL-2572; Manassas, VA, USA) by transduction with lentiviral particles carrying EX-V0108-Lv-225 (FADD cDNA) or its empty control EX-NEG-Lv225 (GeneCopoeia, Rockville, MD), to generate FADD-expressing (JURKAT-FADD) and FADD-deficient (JURKAT-NEG) cell lines as described before [34 (link)]. Cells were grown in RPMI (Thermo Fisher Scientific, Loughborough, UK) supplemented with 15% FBS, 1 mM sodium pyruvate, 2 mM L-glutamine, and 3 µg/mL puromycin. Cultures were maintained at 37 °C in a 5% CO2 humidified atmosphere. Additionally, we corroborated that FADD expression of stable cell lines was equivalent to the FADD endogenous level of the parental clone JURKAT A3 (A3; ATCC, CRL-2570) (Supplementary Figure S1). ATCC routinely performs cell line authentication, using STR profiling as a procedure. Cell experimentation was always performed within a period not exceeding 6 months after resuscitation and in mycoplasma-free culture conditions.

I 2.1 FADD-deficient JURKAT cells were electroporated with EX-V0108-Lv225 or EX-NEG-Lv225 constructs (GeneCopoeia, Rockville, MD, USA) using a Gene Pulser MXcell Electroporation System (Bio-Rad Laboratories, Hercules, CA, USA). 10⁷ cells in 500 μl of complete medium were subjected to 280V, 950 μF, with 5 μg or 25 μg of vector. Electroporated cells were harvested in 10 ml of complete medium and analyzed for protein expression 48 h post transfection, both by flow cytometry - testing GFP positivity with a FACS Canto II (Becton-Dickinson, Franklin Lakes, NJ, USA) for percentage and mean fluorescence intensity - and Western blot. Then, 2 × 10⁵ cells at 10⁶ cells/ml were treated for 24 h with agonist anti-FAS mouse monoclonal antibody (clone CH11; Merck Millipore) or mouse IgM λ isotype control (clone 11E10; Beckman Coulter, Nyon, Switzerland), and aliquoted for (1) protein extraction and WB and for (2) Annexin V/7-AAD apoptosis assay by flow cytometry using PE Annexin V Apoptosis Detection Kit I (BD-Pharmingen™) and a FACSCalibur flow cytometer (Becton-Dickinson). In parallel, untreated transfected cells were followed for cell growth, based on Trypan blue exclusion viability test.