Measuring HIV-1 Infection in CEM.NKR-CCR5 Cells

CEM.NKR-CCR5 cells (9 × 10⁵ cells in 24-well plates with 20 µg/mL of Polybrene) were infected with 200 ng of p24 of luciferase-reporter pseudoviruses, bearing R5-tropic BaL Env, and were produced under the different experimental conditions of this study (see previous point for viral production) in 1 mL of total volume with RPMI 1640 for 2 h (by centrifugation at 1200× g at 25 °C), with subsequent incubation for 4 h at 37 °C, as previously described [12 (link),19 (link),20 (link),21 (link),22 (link),23 (link),25 (link),63 (link),64 (link),65 (link),66 (link),67 (link)]. Unbound viruses were then removed by washing the infected cells. After 24 h of infection, luciferase activity was measured using a luciferase assay kit (Biotium, Hayward, CA, USA) with a microplate reader (VictorTM X5; PerkinElmer, Waltham, MA, USA). Anti-CD4 neutralizing mAb L3T4 (5 μg/mL) was used as a control for the blockade of HIV-1 infection by preincubating permissive CEM.NKR-CCR5 cells with this mAb for 30 min at 37 °C before adding viral input.