Brightglo reagent solution

To evaluate the anti-HIV-1 activity of anti(retro)viral NPs, an in vitro HIV inhibition assay was conducted by infecting TZM-bl cells with HIV-1 pseudovirus (CCR5-using clade A strain Q769.h5) and quantifying the luciferase activity of cell lysates. Briefly, the desired amount of free or encapsulated active agent was dissolved in 1 mL of DMEM, followed by 1:2 serial dilutions to a final volume of 50 μL. Then, 100 μL of cell solution (10⁶ cells per plate) was added to each well. After incubating the cells and treatment for 1 hr at 37°C, 50 μL of virus solution was added to each well. Untreated cells only and virus-infected cells were used as negative and positive controls of infection, respectively. After 48 hr incubation at 37°C, 100 µL culture medium was replaced with 100 µL Bright-Glo reagent solution (Promega Corporation, Madison, WI) and luminescence was measured after 3 min. HIV-1 infection was determined based on the luminescence deviations from the virus-infected cell control.