CD4 T Cell Spinoculation Infection Protocol

CD4 T cells were counted, collected as pellets by centrifugation at 200× g for 10 min at room temperature, and resuspended in the appropriate volume of concentrated viral supernatant. Typically, 50–200 ng of p24^Gag per 4×10⁵ CD4 T cells were used. Spinoculations were performed in 96-well V-bottom plates with up to 5×10⁵ CD4 T cells per well; 15-ml Falcon conical tubes were used for larger quantities of cells (up to 1×10⁷ CD4 T cells/tube). All spinoculations were performed in volumes of 200 µl or less. Cells and virus were centrifuged at 1200× g for 1.5–2 h at room temperature. After spinoculation, cells were pooled and cultured at a concentration of 1×10⁶ cells/ml in RPMI 1640 containing 10% FCS and supplemented with 5 µM saquinavir for 3 days to prevent residual spreading infection. Saquinavir was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH.