LNCaP and PC3 cells were detached using 10 mM EDTA in PBS, washed, resuspended in ice cold FACS buffer and plated in a 96 well plate at 75,000 cells per well. Cells were incubated for 1 hour at 4 °C with JVZ-biotin at different concentrations (25-0.5 µg mL -1 ). For the competition binding assay the concentration of biotin-JVZ007 was kept constant and JVZ007 was added to a molar ratio of 1:1 relative to biotin-JVZ007. After nanobody incubation the cells were spun, washed with FACS buffer and incubated with secondary streptavidin R-phycoerythrin conjugate (SAPE) (BD554061 0.5 mg mL -1 , 500 equivalents relative to biotin-JVZ007) for 1 hour at 4 °C. Cells were then washed, fixed overnight in FACS buffer containing 1% formaldehyde and resuspended in FACS buffer for analysis on a Cytoflex S.