No Nef uptake into Jurkat by flow cytometry.

293T cells were transfected with cDNA3 (Mock), GFP, Nef.GFP, or CD81-GFP plasmid and cultured in exosome-free medium for 3 days. Cell culture media were collected and removed of cell debris (Supernatants), or used to prepare crude exosomes (Exosomes) as described above. Jurkat (1 x 105) were incubated with 100 μl crude exosomes (A) or 100 μl supernatants (B) for 3, 12, 24, or 48 hr. The cells were then washed with PBS multiple times and analyzed by flow cytometry. The data were representative of four independent experiments.