Iodixanol Gradient Ultracentrifugation

PKH26-labeled EVs in 200 μl PBS were loaded on to a 5 ml stepwise gradient of 8, 16, 24, 32, and 40% iodixanol followed by ultracentrifugation at 37,500 rpm (SW55 Ti rotor, Beckman) for 17 h at 4°C. The tube contents were collected from top to bottom into 20 fractions which were assessed for iodixanol concentration using an Abbe refractometer (Bausch and Lomb, Rochester, NY, United States) and PKH26 fluorescence (Ex/Em = 540/585 nm, cut-off = 570 nm) using a Spectra Max M2 spectrophotometer (VWR, Atlanta, GA, United States). The fraction containing the peak PKH26 signal was then assessed for its ability to modulate gene expression in activated mouse HSC.