Optima l 100 k biosafe

The supernatants of infected cells were overlaid on iodixanol gradients formed by equal-volume (2.3-ml) steps of 20, 25, 30, 35, 40, and 45% (wt/vol) iodixanol (Visipaque, 320 mg/ml; GE Healthcare) solutions in phosphate-buffered saline (PBS). Equilibrium was reached by ultracentrifugation for 24 h at 130,000 × g in an SW32.1 Ti rotor at 4°C in a Beckman Optima L-100 K BioSafe ultracentrifuge. Seventeen fractions (1 ml) were collected from the top. The density (g/ml) of each fraction was calculated according to the optical density at 340 nm. AChE activity was measured using Ellman’s method to detect the presence of EVs in each fraction (35 (link)). Briefly, 50 μl of each fraction was incubated at 25°C for 15 min with 150 μl of Ellman solution containing 1 mM acetylthiocholine iodide, 0.23 mM 5,5′-dithiobis(2-nitrobenzoic acid), and 0.45 mM NaHCO₃, all purchased from Sigma. The absorbance was measured at 405 nm. The infectivity of each fraction was assessed as described below.