Fbs complete dmem

TZM-bl cells (a HeLa cell line engineered to express human receptors and coreceptors for HIV, in addition to a Tat-inducible luciferase gene) were used to measure vaccine-induced antibody neutralization of SIV. TZM-bl cells were cultured in T75 flasks (Thermo Scientific) at 10⁴ cell/ml density for 3 d in 10% FBS complete DMEM (GIBCO). On the day of the assay, 1:20 serial fold dilutions of mouse sera were performed. Sera were incubated with SIVmac251.TCLA pseudovirus for 30 min in low-evaporation 96-well clear plates (Corning). TZM-bl cells were detached from flasks using 0.25% trypsin-EDTA (GIBCO) and seeded at a density of 0.5 × 10⁶/ml per well. On the following day, 10% FBS complete DMEM (GIBCO) was added to each well. At day 3, media were aspirated, and cells were lysed using luciferase cell culture lysis buffer (Promega). Luciferase reaction was performed using 30 µl of cell lysis (Promega). The reaction was added to 96-well black optiplates (Perkin Elmer). Luminescence was measured using a Perkin Elmer Victor³ luminometer.