Amicon ultra 50 centrifugal filter units with ultracel 3 membrane 3000mw cutoff membrane

Patients’ adipose tissues and lipoma tissues were minced into small pieces and transferred into a Celstir spinner flask (Wheaton) supplemented with Serum-free Dulbecco’s modified eagle medium/f12 (DMEM/F12) and 1% penicillin/streptomycin, respectively. The tissues were cultured at 37 °C and a rotational speed of 100 rpm for 2 days. The debris of tissues and cells were removed by centrifugation (2000g, 30 min). An additional centrifugation in Amicon® Ultra-50 Centrifugal Filter Units with Ultracel-3 membrane (3000Mw cutoff membrane, Millipore) at 5000g for 30 min was applied to concentrate lipoma tissue extract (LTE) and adipose tissue extract (ATE). Then, the ATE and LTE were mixed with the Total Exosome Isolation™ reagent (Life Technologies) at 4 °C overnight and a final ultracentrifugation step was performed at 10,000g for 1 h at 4 °C. The obtained pellet was resuspended in 400 μl of PBS and stored at − 80 °C with known concentration determined by using the Pierce BCA protein assay kit (KeyGEN, China).