Optima max 130k ultracentrifuge

For the isolation of EVs, cells were grown in 10-cm dishes and conditioned media obtained and cleared by a first centrifugation at 1500 rpm for 5 min at 4°C to eliminate cellular debris. Equal volumes of cleared conditioned media were subjected to ultracentrifugation at 100 000 g (45 000 rpm) in MLS-50 Swinging-Bucket Rotor (Beckman Coulter, cat. no. 367280) mounted on the Optima MAX-130K Ultracentrifuge (Beckman Coulter) for 2 h at 4°C. The floating (S100) and pellet (P100) fractions were then subjected to further analysis.

HSPB1 and huntingtin oligomerization status was analysed by isopycnic ultracentrifugation on a discontinuous sucrose gradient (5–20% w/v), as described by Martin and Ames (116 (link)), with some slight modifications. Briefly, cytoplasmic extracts were prepared with reassembly buffer (100 mM Tris, 0.5-mm MgSO₄, 1-mm EGTA, 2-mm dithiothreitol; pH 6.8), containing protease inhibitors and phosphatase inhibitors (Roche Diagnostics, Mannheim, Germany). Equal amounts of protein were loaded on a 5–20% sucrose gradient and centrifuged at 100 000 g (45 000 rpm) in MLS-50 Swinging-Bucket Rotor (Beckman Coulter, cat. no. 367280) mounted on the Optima MAX-130K Ultracentrifuge (Beckman Coulter) for 24 h at 4°C. Twelve fractions (450 ml each) were collected from the top to the bottom of the gradient. The remaining pellets were resuspended in sample buffer (50 μl) and subjected to immunoblot analysis as above indicated.