Exosome preparation

Exosome preparation

1. Remove media of CDCs at near 100% confluency.
2. Wash cells twice with 10 ml PBS and add 6 ml serum-free Iscove’s Modified Dulbecco’s Media.
3. After 48 hours, harvest the supernatant and centrifuge at 2,500g for 20 minutes.
4. Harvest the supernatant and filter through a 0.22-µm GV filter (Millex) to remove cellular debris.
5. Add 10 ml PBS to equilibrate an Ultra-15 centrifugal filter (10K; Amicon), centrifuge at 4,000g for 10 minutes and aspirate PBS from the filter device and collection tube.
6. Transfer the samples to the filter device and centrifuged at 4,000g for 30 minutes.
7. Evacuate the collection tube, add 10 ml PBS to the filter device and centrifuged at 4,000g for 30 minutes.
8. Recover the samples from the filter device.

References

G. de Couto, R. Gallet, L. Cambier, E. Jaghatspanyan, N. Makkar, J. F. Dawkins, B. P. Berman, E. Marban, Exosomal microRNA transfer into macrophages mediates cellular postconditioning. Circulation 11, 200–214 (2017).