Rantes-PE Binding Assay for CCR5 Expression

First, Rantes-PE was prepared by incubating avidin-PE (Invitrogen, Carlsbad, CA, USA) and biotin-Rantes (Chemotactics) for 3 min at RT. Following this, 1 × 10⁵ cells suspension was incubated with 2U/1 × 10⁵ cells of Heparinise I and III (2U/10⁶ cells, Sigma-Aldrich) for 1 h at 4 °C. Then, cells were washed and incubated with eNAMPT (125 µg/mL; 2.25 µM) or maraviroc (10 µM, Sigma Aldrich) at 4 °C in complete medium (200 µL). After 20 min, Rantes-PE (25 ng; 16 nM) was added to the cell suspension for 2 h at 4 °C. Then, cells were washed three times in ice cold PBS, resuspended in FACS Buffer (Hanks’ Balanced Salt solution HBSS + 0.5% BSA), and samples were analyzed by flow cytometry (BD Accuri C6). eNAMPT was used at this high concentration considering both the molecular weight and the oligomerization state of the proteins, to take into account the reported difference in affinity to CCR5 (0.4 nM for RANTES vs. 5 µM for eNAMPT [12 (link),22 (link)]. In preliminary experiments, lower concentrations of eNAMPT were devoid of effect.