Figure 4

Adaptation of the assay to a multi-well format. (A) Reproducibility of controls. 293T cells transfected with an equimolar mixture of pCHIV and pCHIV^eYFP (wild-type) or their late domain defective variants (late(-)) were seeded into 96-well plate. Fluorescence intensities of tissue culture supernatants (grey bars) or release efficiencies (supernatant fluorescence intensities divided by total fluorescence intensity (supernatant and cell lysate; black bars) were determined as described in methods. Bars represent mean value and standard deviation of 176 wells from 88 plates. (B) Variability across a 96-well plate. 293T cells transfected with an equimolar mixture of pCHIV and pCHIV^eYFP were seeded into 96-well plate. 0.5% of DMSO was added to the medium at 14 h post transfection to mimic the addition of compounds dissolved in DMSO. Supernatants and cell lysates were harvested at 36 h post transfection and relative release efficiencies were determined by dividing extracellular by total fluorescence and normalized by dividing through the median of release efficiencies from all wells of the respective plate. Mean values and standard deviations from three 96-well plates are shown. (C) Variability across a 384-well plate. 293T cells transfected with a mixture of pCHIV/pCHIV^eYFP (black bars) or their late domain defective variants (grey bars), respectively, were seeded into a 384-well plate. Relative fluorescence intensities of the supernatants were measured at 34 h post transfection. Mean values and standard deviations from 24 wells each are shown.