Lentiviral Transduction of Activated Mouse CD4+ T Cells

On day 1, HEK293T cells were transfected with 1 μg of vector plus 0.5 μg of pCL-Eco plasmid (Naviaux et al., 1996 (link)) using X-tremeGene 9 DNA transfection reagent (Roche). On day 2, the media was exchanged with 1 ml fresh T cell media. CD4⁺ T cells were isolated from the spleens of B6 mice using an EasySep Mouse Naive CD4⁺ T cell isolation kit (Stemcell Technologies) and cultured in 96-well plates coated with anti-CD3 antibody (10 μg/ml; BD Biosciences) at a density of 1–2 × 10⁶ cells per well in 0.1 ml of T cell media with soluble anti-CD28 antibody (10 μg/ml; BD Biosciences). On day 3, the activated T cells were transferred to 24-well plates at a density of 1–2 × 10⁶ cells per well, washed, and then resuspended in virus-containing supernatants from the HEK293T cell cultures. Polybrene was added to a final concentration of 8 μg/ml. The cell–virus mixture was then centrifuged at 3,500 rpm for 90 min at 32°C to achieve transduction. Following transduction, cells were cultured in 96-well plates coated with anti-CD3 antibody (1 μg/ml) at a density of 5 × 10⁵ cells per well in T cell media with soluble anti-CD28 antibody (1 μg/ml). On the following day, cells were washed and cultured in fresh 96-well plates in T cell media with IL-2 (20 ng/ml) until ready for further analysis.