I am currently working on extracellular vesicles (EVs) and I intend to perform uptake/internalization assay using SH-SY5Y neuroblastoma cell line. I experimented on the PKH26 dye which could be used to stain cell membrane as well as EVs. Based on published literatures, there are several methods that have been used to remove unbound dye after EVs labelling which includes ultracentrifugation, ultrafiltration using the viva spin column filters, sucrose density gradient based isolation. I tweaked existing protocols and I stained my EVs and removed unbound dyes by performing several wash steps and concentrating my samples in the 100KDa ultra centrifugal filter device. As a control I stained PBS with the PKH dye and co-cultured both PKH-26 labeled EVs and PKH-26 labelled PBS (control) with SH-SY5Y cells for 24 hours.

Using the fluorescent microscope, I could not distinguish the true signals as the control (PKH+PBS) (LOWER IMAGE ATTACHED )has the same fluorescent signal like the PKH-labelled EVs (UPPER IMAGE ATTACHED).

What could be responsible for this?