Beckman l90k

HN-EVs were isolated from spider venom using density gradient centrifugation as previously described (Figure 2A), with minor modifications [19 (link),48 (link)]. Briefly, the venom was centrifuged at 10,000× g for 5 min to remove glandular endothelial cells and other cell debris. The supernatant was layered on preformed 10–50% (w/v) Nycodenz (Sigma Aldrich, St. Louis, MO, USA) gradients in PBS in 12.5 mL Ultra-Clear tubes. First, the tubes were centrifuged at 100,000× g for 120 min at 4 °C in an SW41Ti rotor in a Beckman L90K centrifuge (Beckman Coulter, Woerden, Netherlands). Then, the light-scattering band was washed with PBS and centrifuged at 100,000× g for 70 min. Finally, the pellet (which contained EVs) was resuspended in 100 μL PBS. The protein concentration was determined by the BCA protein assay kit (Thermo Fisher, Waltham, MA, USA). The EVs were either used right away or stored at −80 °C for later use.

MP was prepared as previously reported with some modifications [21 (link)]. Briefly, the longissimus dorsi of pork was quickly cut into small pieces and then ground two times at 2000 r/min for 10 s through a Waring Blender (GM200, Restch, Haan, Germany). MP was extracted with four volumes of cooling isolation buffer (10 mM Na₂HPO₄/NaH₂PO₄, 0.1 M NaCl, 2 mM MgCl₂, 1 mM EGTA, pH 7.0, 4 °C) and centrifugated (Beckman L-90K, Beckman, Pasadena, CA, USA) at 3000× g for 15 min at 4 °C. The supernatant was poured out, and the pellets were re-suspended with a homogenizer (Ultra Turrax T-25 BASIC, IKA Company, Staufen, Germany) and centrifuged twice more under the same conditions. Then, the pellet was mixed in four volumes of salt solution (0.1 M NaCl), centrifuged (3000× g for 15 min), and washed three times. Before the final wash, the MP was filtered with three layers of gauze. The final pellet collected was MP, stored at 4 °C for further use within 48 h.