Experimental plan and fluorescence images. (A) Scheme of the experimental plan. (B) SDS-PAGE analysis of ATL. Proteins gel electrophoresis separation was run on acrylamide gradient (4%-20%). The ATL sample is shown after Coomassie brilliant Blue staining of the proteins bands (lane 1) and by UV transillumination, before staining of the proteins bands, to reveal the extent of the fluorescence labelling (lane 2). (C) FRET analysis of mDC loaded with Alexa488-labeled ATL and immunolabelled with HLA-DR(HL12) mAb and Alexa546-conjugated IgG. The upper panels refer to the sample analyzed at 16 hours after the maturation stimulus and the lower panels refer to the sample analyzed at 46 hours after the maturation stimulus. Panels are divided in sets of images acquired before and after acceptor photobleaching (see Materials and Methods). Donor images were acquired in the green channel (a, h, d and k) and acceptor images were acquired in the red channel (b, i, e and l). White arrows (e and l) indicate the bleached regions. The relative merged images are also shown (c, j, f and m). FRET efficiency was calculated using eq. 1 and the results are presented as pseudo-color images (g and n).