No Nef uptake into 293T by microscopic imaging.

293T (2 x 106) were plated at a 10 cm plate, transfected with GFP, CD81.GFP, or Nef.GFP plasmid, and cultured for 16 hr, followed by direct microscopic imaging with a FITC filter or under the bright field (a 10X objective) (A). Transfected cells were then cultured in exosome-free medium for 3 days. Crude exosomes from the culture medium (40 ml) were prepared as described above, suspended in exosome-free medium, added onto fresh 293T in a polylysine-treated glass bottom dish, and incubated for 3 hr (B) and 12 hr (C). At the end of each incubation, images of the target cells were taken with a FITC filter or under the bright field (a 100X objective). The micrographs were representative of images from multiple fields of two independent experiments.