Lentiviral particles were made in a biosafety level 2 laboratory by transfecting a prolentiviral construct and packaging and coat pseudotyping DNA constructs (pCAG-SIVgprre, pCAG4-RTR-SIV, pCMV-VSV-G)51,53 into HEK293T/17 cells (ATCC) in 10-cm plates. After 72 h, supernatant was collected (6 mL) and filtered. Neuronal cultures were infected at 3 days in vitro. Each well of a 24-well plate was incubated overnight with 0.5 mL of lentivirus in conditioned growth medium. The growth medium was supplemented with 4 μM AraC to inhibit glial proliferation. In some experiments, OGB1-AM was loaded into cells by incubating neurons in 1 mL of 2 μM OGB1-AM (Invitrogen) for 30 min and rinsing 3 times with imaging buffer (145 mM NaCl, 2.5 mM KCl, 10 mM glucose, 10 mM HEPES pH 7.4, 2 mM CaCl2, 1 mM MgCl2).
