Dual Chamber Transmission Assay for HIV-1

Dual chamber transmission assay was performed as previously described [28 (link)]. HeLa cells were seeded into an upper chamber of a transwell plate (Sigma-Aldrich, Liège, Belgium), while TZM-bl cells were seeded in the lower chamber. Trans Epithelial Electric Resistance (TEER) was measured using Millicell-ERS Volt-Ohm Meter. HIV-1 ADA-M (200 pg) and drugs were added to the upper chamber after 4 days when TEER reached 150 Ohm/cm². 24 h after infection luciferase value of TZM-bl cells lysate was measured using the Luciferase System Kit and the POLARstar Omega Plate Reader. Data were analyzed using GraphPrism. The HIV-1 DC-SIGN transmission assay was performed as previously described [27 (link)]. To investigate the cellular activation induced by CE or piceatannol, CD25 and CD69 expression was measured on PBMCs after incubation with CE/piceatannol or 10 µg/mL PHA-P for 24 h at 37 °C using FITC-conjugated anti-CD4, PE/Cy7-conjugated anti-CD25, PE-conjugated anti-CD69 mAbs (Biolegend, Amsterdam, Netherlands), and the near-IR fluorescent reactive dye.