Figure 3: DCs infected with HIV-1 release exosomes and virions having distinctive markers and sedimentation velocities. DCs were incubated for 1h with mock viral suspension (open bars) or with exosome depleted suspension of NL4-3Balenv produced on PBMCs (black bars) and then cultured for an additional 48h. Cellfree supernatants were subjected to differential centrifugations to pellet exosomes and HIV-1. Exosomes were then separated from virions using Optiprep™ gradient separation. Exosomes in the 100,000xg pellet (a) and in each individual Optiprep fraction (b) were quantified by measuring AChE activity. The virions were quantified using an anti-p24gag ELISA (c). Data correspond to the mean ± SEM of triplicate samples from one donor and these results are representative of three independent donors. ** P < 0.01. The infectivity of viruses present in each gradient fraction was evaluated by means of real-time PCR targeting the spliced TAT gene in autologous T CD4+ lymphocytes (d).