From: Are exosomes the vehicle for protein aggregate propagation in neurodegenerative diseases?
1. Differential ultracentrifugation |
Culture supernatant or fluid – at each of steps, the supernatant is used for following step. |
⇒ 300 x g, 10 min (→ Pellet: cells) ⇒ 2000 x g, 10 min (→ Pellet: dead cells) ⇒ 10,000 x g, 30 min (→ Pellet: cell debris) ⇒ Final 100,000 x g, 70 min → Pellet: exosomes + contaminating proteins ⇒ Wash in PBS 100,000 x g, 70 min → Pellet: correspond to exosomes |
2. Rate-zonal centrifugation |
- Load exosome preparation at the gradients (0.25 - 2 M sucrose) and centrifuge overnight (≥14 h) at 210,000 x g, 4 °C. - With a micropipettor, collect eleven 1-mL fractions, from top to bottom. |