Time course of labelled VLP release cells determined by fluorescence measurement or ELISA, respectively. 293T cells were transfected with mixtures of pCHIV/pCHIVeYFP (filled circles) or pCHIVlate(-)/pCHIVeYFPlate(-) (open circles), respectively. Cells transfected with peYFP-C1/empty vector (open triangles) or peYFP/pCHIV (filled triangles) served as controls. At the indicated time points after transfection, tissue culture supernatants were sampled and analyzed in parallel by fluorescence measurements (A) or ELISA (B), respectively, as described in methods. To control for transfection efficiency, cell lysates prepared at the end of the experiment were subjected to the same analyses. Normalized release efficiency was determined by subtracting the background derived from untransfected cells and dividing extracellular fluorescence or p24 CA versus intracellular fluorescence or p24 CA determined at 48 h post transfection, respectively. Data represent mean values and standard deviations from triplicate transfections. (C) Increase of unspecific release upon prolonged incubation of cells. Normalized release values for HIVlate(-)/HIVeYFPlate(-) determined in the experiment shown in Figure 3A at the indicated times post transfection were divided by the respective values obtained for HIV/HIVeYFP.