Box 1. RetroNectin treatment for colocalization of virus and target cells

RetroNectin may be used to facilitate colocalization of virus and target cells. This is particularly important for retrovirus transduction⁹ but can generally be omitted when using Lentivirus/VSV pseudotyped virus. It is included here should readers elect to use retroviruses for rapidly dividing cell targets.

Infection of PBLs:

1. On Day 3 after lymphocye stimulation with anti-CD3 and anti-CD28, coat a 24-well plate with 1 ml per well of RetroNectin (12 g ml^-1).
2. Incubate at 4 °C overnight.
3. On Day 4 after lymphocyte stimulation, remove the RetroNectin and wash with PBS once.
4. Block plate with 1% BSA in PBS at 37 °C for 20 min.
5. Remove BSA and wash once with PBS.
6. To each well add 0.5^-1 10⁶ lymphocytes per ml in 2.8 ml of filtered PT67 supernatant.
7. Culture for 4 d at 37 °C in a 5% CO₂ atmosphere.
8. Remove supernatant from log-phase PT67 packaging cells infected with GFP-chTCR retroviral vector.
9. Pass supernatant through a 0.45 m filter.
10. Incubate the culture at 37 °C for 5 h; gently remove viral supernatant.
11. Replace with RPMI-GM containing 50 ml^-1 of IL-2.
12. Repeat the transduction procedure next day.
13. Harvest lymphocytes after 2 d by vigorous flushing and washing of the wells.
14. Resuspend cells in RPMI-GM containing 50 U ml^-1 IL-2 and incubate in a 5% CO₂ atmosphere at 37 °C.
15. Add additional IL-2 (50 U ml^-1) every other day.
16. After 2–4 d, begin analyzing the cells daily for GFP expression using an inverted fluorescence microscope.