Biotek synergy 4 hybrid microplate reader

To test the percentage of inhibition due to the interaction between the G2-S16 dendrimer and RSV, viral particles were pre-incubated at a MOI 3 with G2-S16 dendrimer 1 h at 4 °C. HEp-2 cells were plated in a 96-well plate in DMEM medium at 2% FBS and infected with the RSV-G2 inoculums for 1 h at 4 °C. Cells were washed twice to remove unbound virus and fixed with 4% formaldehyde and blocked with 5% SAB in PBS-Tween. Cell-bound virus was determined by incubating a primary antibody (1:50 in PBS-Tween) for 1 h at RT. Secondary antibody (anti-mouse–IgG peroxidase, 1:500 in PBS-Tween) was added after washing the plates twice, and it was incubated for 2 h at 37 °C. Then, the secondary antibody was removed, plates were washed, and ABTS developing substrate (2,2′-azino-bis- (3-ethylbenzothiazoline)-6-sulfonic acid)) (Thermo Scientific, Rock-ford, IL, USA) was added and incubated for 20 min at RT. The reaction was stopped with 1% sodium dodecyl sulfate (SDS) stop solution, and the absorbance at 405–410 nm was measured on a microplate reader BioTek Synergy™ 4 Hybrid Microplate Reader (BioTek, Winooski, VT, USA).

In order to determine the R5-HIV-1_NLAD8 quantity, each supernatant and cell pellet was analyzed by enzyme immunoassay for the quantitative detection of HIV-1 p24 antigen (INNOTEST HIV Antigen mAb, FUJIREBIO, Ghent, Belgium) following the manufacturer’s instructions. Supernatants and pellets were incubated in a 96-well plate coated with p24 antigen, and the absorbance was measured by the presence of viral protein HIV-1 p24 at 450 nm in a microplate reader BioTek Synergy™ 4 Hybrid Microplate Reader (BioTek, Winooski, NA, USA).