HIV-1 infection assay in TZM-bl cell line

The TZM-bl indicator cell line (obtained through NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: TZM-bl from Dr John C Kappes, Dr Xiaoyun Wu and Tranzyme Inc. (Research Triangle Park, NC, USA))^22,23,24,25 was maintained in DMEM supplemented with 10% heat-inactivated FBS (Invitrogen). The cell line was used for quantitative analysis of HIV infection using luciferase as a reporter.

Briefly, 60 000 TZM-bl cells were seeded into a 24-well plate in 500 μl of 10% DMEM. Various treatments containing either media alone or recombinant IFNβ (100 pg/ml) or GEC supernatants from mock control or gp120-treated monolayers incubated in the presence or absence of rabbit anti-human IFNβ (AB1431; Millipore, Etobicoke, ON, Canada) or rabbit isotype IgG control (ab172730; Abcam Inc., Toronto, ON, Canada) at 10 μg/ ml concentrations were added to TZM-bl cells in a total volume of 250 μl. Each sample was added to TZM-bl cells in the presence of HIV-1 for 2 h with a gentle shaking every 15 min. After 2 h, 1 ml of 10% DMEM was overlaid and the cells were further incubated for 24 h at 37 °C. After 24 h, the cells were washed once with 1 × phosphate-buffered saline, lysed and the luciferase activity was measured using a Stratagene luciferase assay kit (Agilent Technologies, Mississauga, ON, Canada) as per the manufacturer’s instructions.