Retroviral transduction of primary T cells

The coding region of the S1P reporter was cloned into the pMXs retroviral vector. pCL-Eco Retrovirus packaging vector (Novus Biologicals) and HEK293T cells were used for generation of retrovirus. T cells were purified from lymph nodes and spleen by negative selection with magnetic beads (EasySep™ Mouse Biotin Positive Selection Kit from StemCell technologies or MagniSort™ Streptavidin Negative Selection Beads from eBioscience) using biotinylated antibodies against CD19 (6D5), CD11c (N418), and NKp46 (29A1.4). Purified T cells were resuspended in activation media containing 1 μg/ml anti-CD28 (37.51) and 0.25 μg/ml anti-CD3 (145-2C11), and plated on anti-hamster IgG (MP Biomedicals) coated plates overnight. T cells were transduced the following day with the retrovirus encoding the S1P reporter by spinning at 1258g (no brake), 90 min, 32° C with polybrene (8 μg/ml); incubated at 37°C overnight with new activation media; and transduced a second time the following day. T cells were rested for two days without anti-CD28 and anti-CD3, and transferred i.v. into wild-type mice. LN from recipient mice were analyzed the following day by confocal microscopy.