Figure 4.

BlaM HIV-1 fusion quantification per cell in primary T cells. (A) Micrographs for a population of primary T cells exposed to HIV-1_HXB2 virions and their corresponding pixel-by-pixel histograms are also presented for FRET-intensity-based and f_D methods. In all cases, No Env HIV-1 virions were employed as a negative control and the CCF2-AM basal cleavage utilized as a reference for negative fusion. The images are pseudocolored (red, fusion negative; green, fusion positive). Scale Bars: 20 µm (B) The normalized statistics for a population of cells with single cell accuracy are presented for the methods outlined above; n > 240 cells for each condition. Results are from one experiment with cells from one donor. Error bars are the standard deviation (SD) of the mean. (C) The overall percentage of fusion for a population of TZM-bl cells exposed to HIV-1_HXB2 virions is presented for both methods. An average of ~5% was found for intensity-based and f_D. (D) Using each calibration curve, the relative amount of fused HIV-1_HXB2 virions per T cell was obtained, again with single cell accuracy. Images are pseudocolored (cold colors represent relatively low amount of internalized viral particles and warm colors, relatively high amount of internalized particles). The frequency plots per cell are plotted for each approach (right column).