Optipreptm density gradient medium

Binary mixtures of fluorescent polystyrene particles of 30.2 μm and 20.4 μm diameter (SPHERO^TM Fluorescent particles, FP-30052-5 and FP-20056-5, Spherotech, Inc.) were used for the segregation experiments. For multiplexed segregation, particles of 10.1, 20.4, and 30.2 μm diameter were used. The focusing and splitting experiments used a solution of 30.2 μm particles. The particles were re-suspended in a density-equilibrated buffer that is a mixed solution of deionized water, OptiPrep^TM density gradient medium (Sigma-Aldrich Co.), and Tween 20 surfactant of 0.1% v/v (Sigma-Aldrich Co.). The number of the particles in the array was adjusted by varying the concentration of the particles in the suspension. The solution exchange experiments used two solutions including the density-equilibrated buffer and a fluorescein solution (0.004 g/mL, fluorescein sodium salt dissolved in the density equilibrated buffer) (Sigma-Aldrich, Co.). Before the experiment, the particles of 30.2 μm diameter were re-suspended in the fluorescein solution. Once the fluorescein solution was loaded into the device filled with the density-equilibrated buffer, the oscillatory flow displaced the particles from the fluorescein solution to the buffer. The fluorescence of the array region was measured before and after the segregation.

Flotation in an iodixanol gradient was performed as described in Crescitelli et al. (2020 (link)) with some modifications. The 100K pellet obtained after differential centrifugation was re‐suspended in 1 mL of 40% iodixanol (v/v) (OptiPrep^TM Density Gradient Medium, Sigma) in PBS and bottom loaded. For the discontinuous iodixanol gradient equal volumes of solutions of 30%, 20% and 10% iodixanol were layered on the top of the sample (Figure 6a) and centrifuged in a 4‐mL tube (Beckman Coulter, 328874) at 180,000 × g for 19h, at 0°C (Sorvall WX‐Ultra 80, Thermo Fisher Scientific) in a TH‐660 rotor (Thermo Fisher Scientific). Fractions of 500 μL from top to bottom were collected from the tube. An opaque band of EVs was recovered in fraction 6.

Identification of lipid rafts through flotation on OptiprepTMsucrose gradient is a standard procedure, widely described in the literature^{44 (link)–46 (link),63 (link)}. Here we followed the protocol used to study the association of PRV US9 with lipid rafts from infected cells^{43 (link)}.
Briefly, cells were harvested, washed once on ice in PBS, lysed in 1 ml ice cold lysis buffer (1% Triton X100 in TNE: 25 mM Tris pH 6.8, 150 mM NaCl, 5 mM EDTA) with proteases/phosphatases inhibitors, homogenized by passing 15 times through an 18-gauge needle, rocked for 30′ at 4 °C, homogenized again (5 times through an 18-gauge needle), and finally mixed with 2 ml of ice-cold 60% OptiprepTM density gradient medium (Sigma-Aldrich). The gradient was prepared by placing the cells homogenate at the bottom of a Beckman SW41 ultracentrifuge tube and subsequently overlaying it with 5 ml of ice-cold 30% Optiprep in TNE and 4 ml of ice-cold 5% Optiprep in TNE. Samples were centrifuged at 34,200 rpm (200,000 × g) at 4 °C for 20 hours. 1 ml fractions were collected from top and analyzed by SDS Page.