Isolation and Characterization of Microvesicles from HIV-Infected Macrophages and LPS-Activated Microglia

MVs were isolated from the supernatants of HIV-1-infected macrophages and LPS-activated microglia through differential centrifugations with or without neutral sphingomyelinases inhibitor GW4869 (Sigma) at different dosages, 2, 5 and 10 μM. Briefly, the supernatants were first centrifuged at 300 × g for 10 min to remove free cells, at 3000 × g for 20 min to remove cellular debris and then 10,000 × g for 30 min to remove free organelles. Lastly, MVs were collected by ultracentrifugation at 100,000 × g for 2 h at 4 °C. To prepare MVs for Western blot, the MVs pellets were lysed in M-PER mammalian protein extraction reagent (Thermo Scientific, Pittsburgh, PA). For negative staining, MVs were fixed in 2 % glutaraldehyde and 2 % paraformaldehyde. For glutaminase activity assay and neurotoxicity, the MVs were resuspended in 1 ml of glutamine-free neurobasal medium.