Alliance p24 elisa kit

Untreated cells and cells treated for 1, 2, or 3 days with CM were harvested with cellstriper (Mediatech) and seeded in 12- well plates with a cell density of 0.2 × 10⁶ cells/ml (1 ml/well). Untreated cells were seeded either in fresh medium or in 1:2 diluted CM. Cells were then infected with 4 μl/ml of HIV-1 strains IIIB or BaL (10⁵ TCID₅₀ per μL; Advanced Biotechnologies, Inc., Columbia, MD) per well. Two hours after infection, cells were washed with PBS, and reseeded in 12-well plates. Untreated cells that were infected in the presence of CM were either seeded in fresh or 1:2 diluted CM. Twenty-four hours after the infection, the medium from each well was collected and cells were removed by centrifugation at 1100 rpm for 5 min. The supernatant was collected and HIV-1 p24 was quantified using an Alliance p24 ELISA kit as described by the manufacturer (PerkinElmer, Wellesley, MA).