Transduction of TAT-FITC assayed by flow cytometry and confocal microscopy. Transduction of TAT-NBD and TAT-Ctrl peptides was studied by flow cytometry (A) and confocal microscopy (B-D). (A) Isolated neutrophils were incubated with buffer, free FITC, FITC-labeled TAT-Ctrl, or FITC-labeled TAT-NBD (5 μm) for 20 minutes at 37°C. Flow cytometry shows that both the FITC-labeled TAT-NBD and the TAT-Ctrl peptides stained almost 100% of the cells. For confocal microscopy, live cells were treated with the indicated peptides and control FITC (green; Bi,Ci,Di), washed once, and incubated in the live-cell DNA dye DRAQ5 (red; Bii,Cii,Dii). Samples were immediately analyzed by confocal microscopy without fixation. The images demonstrate that TAT-NBD (C) and TAT-Ctrl (D) localize throughout the cytoplasm and the nucleus, whereas free FITC (B) does not transduce into cells. In each case, an overlay image is shown at the bottom right-hand side (Biv,Civ,Div). To better visualize the localization of the TAT-Ctrl and TAT-NBD, only the green and red channels were overlaid. TAT peptides are taken up into the cytoplasm and nucleus and accumulate to a lower extent in chromatin dense areas. Scale bars correspond to 50 μm in the upper panels (i-ii) and 10 μm in the lower panels (iii-iv).