Cy3 fast conjugation kit

Two approaches were used to incorporate ARV into HIV-negative BE. Emtricitabine (50 μg FTC) (NIH AIDS Reagent Program) was Cy3-labeled with Cy3 Fast Conjugation kit (Abcam, Cambridge, UK) according to manufacturer’s instructions. Modifier Reagent (1 μl per 10 μl FTC) was mixed with Cy3 Conjugate. Cy3 Quencher reagent (1 μl per 10 μl FTC) stopped the reaction. First, HIV-negative BE (200 μg) were combined with 50 μg FTC-Cy3 or buffer control for 90 minutes at 37°C before ultracentrifugation at 100,000 × g for 70 minutes. Pellets containing FTC-Cy3 loaded or unlabeled FTC control EVs were resuspended in PBS to the original volume and protein was quantified by Nanodrop absorbance before Cy3 detection and HIV inhibition studies^{19 (link)}. Alternatively, 200 μg HIV-negative BE in 50 μl volume were incubated with 50 μg FTC-Cy3 in 20 μl, 10 μl ExoFect reagent (SBI), and 70 μl PBS. Following 10 minute incubation at 37°C, ExoQuick reagent (30 μl) was added. The transfection/ExoQuick solution was placed on ice for 30 minutes prior to centrifugation (13,000 rpm, 3 minutes). Pelleted EVs were resuspended in the original volume of PBS before quantification, Cy3 detection, and HIV inhibition studies^{20 (link)}. Both approaches were assessed in three independent donors. 100 μg/ml EVs were used for HIV inhibition studies.