4.4. HIV-1-Mediated Cell–Cell Fusion Assay

A dye transfer assay was used to detect HIV-1 Env-mediated cell-cell fusion as described previously [50]. One mL of 2 × 10⁴/mL H9/HIV-1IIIB cells labeled with 2.5 μL of 1 nM fluorescent reagent, Calcein AM (Molecular Probes, Inc., Eugene, Oregon), was incubated at 37 °C for 30 min. For each well of the 96-well plate, 50 μL 2 × 10⁴/mL labeled H9/HIV-1IIIB cells were then incubated with 100 μL 1 × 10⁵/mL MT-2 cells at 37 °C for 2 h in the presence or absence of the tested peptide at graded concentrations. The fused and unfused Calcein-labeled HIV-1 IIIB cells were counted under an inverted fluorescence microscope (Zeiss, Oberkochen, Germany). The IC₅₀ values were calculated by using the Calcusyn computer program (Biosoft, Ferguson, MO, USA).

An HIV-1 Env-mediated cell-cell fusion washout assay was conducted as previously described [51]. Briefly, HP23-E6-IDL (5 nM), sCD4 (100 nM), and HP23-E6-IDL/sCD4 mixture, respectively, were pre-incubated with H9/HIV-1IIIB cells labeled with Calcein AM at 37 °C for 0.5 h. The treated H9/HIV-1IIIB cells were washed with PBS (unwashed H9/HIV-1IIIB cells with HP23-E6-IDL were taken as control) before addition of MT-2 cells. After incubation at 37 °C for 2 h, the fused and unfused Calcein-labeled HIV-1 IIIB cells were counted as described above.