Nucleolin Surface Binding Assay

ssON’s interaction with nucleolin expressed on the surface of cells was assessed using a modified version of the flow cytometry-based cell surface binding assay (9 (link)). A549 cells were detached with 2mM EDTA, chilled on ice and pre-incubated with ssON (1 µM) for 30 min at 4°C, followed by incubation with anti-nucleolin antibody (Ab22758, Abcam) at different concentrations for 1h at 4°C and then washed. The binding of the anti-nucleolin antibody to the cell surface, was revealed by staining with Alexa 488-conjugated donkey anti-rabbit IgG secondary antibody (5 µg/ml; Invitrogen) for 1h at RT. Cells were then washed and the data acquisitions were obtained using a FACSVerse (BD) and all analyses were performed with FlowJo software (Tree Star).