1. Ultracentrifuge the virus at 50,000 x g for 90 min at 4°C.

Remove the supernatant. Drain carefully and well (preferably with a pipet) since the viral pellet is glassy and will be a filmy smear on the side of the tube.

2. Resuspend the virus to 0.5–1% of the original volume in TNE and incubate overnight at 4oC. Swirling during incubation may damage the virus. Gently pipet the solution to mix only after the overnight incubation, to allow diffusion of the virus. TNE is Tris buffer with NaCl and EDTA, which helps maintain the pH and is appropriate for storage if desired. Media can be used if the virus will be used immediately.

3. If desired, perform a second round of ultracentrifugation (Steps 1–2) by pooling previously concentrated virus.

4. This step is necessary only if injecting into animals, not if infecting cells in culture: Remove cellular debris and aggregated virus by low speed centrifugation (500 x g) for 5 min at 4oC.

5. Determine the viral titers of pre- and post-concentrated viral supernatants.

6. Infect target cells according to the Retroviral Gene Transfer and Expression