Mls50 tubes

The stored, healthy, cell-free SF of 2 donors (adult horses; 6 ml/donor) was thawed, pooled, and divided into 6 aliquots of 2 ml. Each aliquot was incubated with HYase and cleared from protein aggregates as described above. Supernatants were pooled, transferred into MLS50 tubes (Beckman-Coulter) (3 ml per tube) and gently mixed with 2 ml PBS. EVs were pelleted with 2 sequential ultracentrifugation steps of 10,000g (35 min, 10,000 rpm; RCF average 8,025g; RCF max 10,730g; κ-factor 1,777) and 200,000g (120 min, 50,000 rpm; RCF average 200,620g; RCF max 268,240g; κ-factor 71.7) using an MLS50 rotor in a Beckman-Coulter Optima™ MAX-E ultracentrifuge at 4°C. Note that by omitting the 100,000g centrifugation step, all EVs that would have been recovered in a separate 100,000g step are now recovered in the 200,000g step (in this paper these vesicles will be referred to as “100/200,000g EVs”). The EV pellets of each of 4 corresponding tubes were resuspended in PBS, pooled (final volume 250 µl EVs), and mixed with 1.25 ml 2 M sucrose solution (in PBS) and used for sucrose density gradient floatation.

Pelleted 10,000g or 100/200,000g EVs, mixed with 2 M sucrose solution (total volume=1.5 ml) in MLS50 tubes (Beckman-Coulter) were carefully overlaid with sucrose solutions of 1.4, 0.4, and 0 M (sucrose in PBS) to create 2 discontinuous sucrose gradients (Supplementary Fig. 1). Gradients were centrifuged at 200,000g for 16 h at 4°C (Beckman-Coulter Optima™ MAX-E centrifuge; MLS50 rotor; 50,000 rpm; RCF average 200,620g; RCF max 268,240g; κ-factor 71.1). After centrifugation 5 fractions of 1 ml were collected from bottom (fraction 5) to top (fraction 1) by using a peristaltic pump connected to a capillary tube reaching to the bottom of the centrifuge tube. Fraction densities were calculated by refractometry.