Fitc conjugated goat anti rhesus igg h l polyclonal antiserum

Indirect surface staining was used to measure the ability of plasma samples to bind HIV-1 envelope expressed on the surface of infected cells⁷² . CEM.NKR_CCR5 cells were mock infected or infected with HIV-1 infectious molecular clones⁷³ expressing the 1086.C or CH505.TF envelope proteins. The cells were incubated with a 1:100 dilution of plasma samples for 2 h at 37 °C and then stained with Live/Dead Aqua Dead Cell Stain (Thermo, Fisher Scientific, Waltham, MA) to exclude dead cells from analysis. Cells were washed and then permeabilized with Cytofix/Cytoperm solution (BD Biosciences, San Jose, CA) prior to staining with fluorescein isothiocyanate (FITC)-conjugated goat anti-rhesus IgG(H + L) polyclonal antiserum (Southern Biotech, Birmingham, AL, Cat# 6200-02) and RD1-conjugated anti-p24 MAb KC57 (Beckman Coulter, Inc., Indianapolis, IN, Cat# 6604667). Cells positive for plasma binding were defined as viable, p24 positive, and FITC positive. Final results are reported as the FITC MFI of the live infected cell population (p24-positive cells) after subtraction of the background observed for the pre-infection (week 0) samples.

Indirect surface staining was used to measure the ability of plasma samples to bind the HIV-1 envelope expressed on the infected-cell surface by methods similar to those previously described (80 (link)). CEM.NKR_CCR5 cells were mock infected or infected with an HIV-1 infectious molecular clone (81 (link)) expressing the 1086.C or SHIV1157ipd3N4 envelope protein. The cells were incubated with a 1:100 dilution of plasma samples for 2 h at 37°C and then stained with Live/Dead Aqua Dead Cell Stain (Thermo, Fisher Scientific, Waltham, MA) to exclude dead cells from analysis. Cells were washed and then permeabilized with Cytofix/Cytoperm solution (BD Biosciences, San Jose, CA) prior to staining with fluorescein isothiocyanate (FITC)-conjugated goat anti-rhesus IgG (H+L) polyclonal antiserum (Southern Biotech, Birmingham, AL) and RD1-conjugated anti-p24 MAb KC57 (Beckman Coulter, Inc., Indianapolis, IN). Cells positive for plasma binding were defined as viable, p24/27 positive, and FITC positive. Final results are reported as the FITC MFI of the p24/27-positive events after subtraction of the background observed for the matched maternal prevaccination samples.