MTT assay

The MTT assay to determine cytotoxicity (50% inhibitory concentration [IC₅₀]), cytoprotective (antiviral) effect (50% effective concentration [EC₅₀]) and selectivity index (SI) was performed in triplicate as previously described.53 Briefly, MT4 lymphocytes were seeded in wells of a microtiter plate at 6×10⁵ cells/mL and treated with serial dilutions of azidothymidine (AZT), dendrimer alone, dendriplex with control RNA or dendriplex with Ψ RNA. A total of 50 µL of RPMI 1640 medium was added to each well in one half of the plate (mock infection), while 50 µL of HIV_IIIB at 300 tissue culture infective dose (TCID)₅₀ was added to the other half (HIV infection). The plate was incubated for 5 days at 37°C in 5% CO₂. At the end of the incubation period, the MTT assay was performed to assess toxicity in mock (uninfected) cells and cytoprotective effect in HIV-infected cells. The optical density readings were plotted on a graph, and the IC₅₀ and EC₅₀ were determined by linear extrapolation. Untreated, uninfected cells and untreated, HIV-infected cells were deemed to have 100% and 0% viability, respectively. Typically, the viability of the untreated, uninfected cells, as measured by the optical density reading, was at least fivefold that of untreated, HIV-infected cells.53 Growth medium without phenol red was used in the assay to improve sensitivity of absorbance readings.