Sw 32 ti rotor swinging bucket rotor

iMSC-derived lEVs and sEVs were isolated by serial centrifugation with ultracentrifugation according to previous described methods [14 ,21 ]. Briefly, the iMSC culture medium was collected and centrifuged at 300×g (4 °C) for 10 min to remove dead cells. Then, the supernatant was centrifuged at 2000×g (4 °C) for 20 min to remove apoptotic bodies and cell debris. Next, the supernatant was centrifuged at 10,000×g (4 °C) for 30 min to pellet the lEVs. Afterwards, the lEVs-depleted supernatant was filtered through a 0.22 μm disposable membrane (Millipore, US), and further ultracentrifuged at 100,000×g (4 °C) for 70 min using an SW 32 Ti Rotor Swinging Bucket rotor (Beckman Coulter, Fullerton, CA) to pellet the sEVs. The lEVs pellet was re-suspended in a large volume of PBS followed by 10,000×g (4 °C) centrifugation for 30 min to wash the sample. Likewise, the sEVs pellet was re-suspended in a large volume of PBS followed by 100,000×g (4 °C) ultracentrifugation for 70 min to wash the sample. After PBS-washing, the lEVs and sEVs were respectively re-suspended in sterile PBS and stored at −80 °C for up to 1 month before use.

iMSC-sEVs were isolated from iMSC-conditioned medium (iMSC-CM) by differential ultracentrifugation, as described previously (Hu et al., 2020). Briefly, iMSC-CM was centrifuged at 300 × g for 10 minutes, 2000 × g for 20 minutes, and 10,000 × g for 30 minutes, respectively. The supernatant was then filtered through a 0.22 µm sterilized filter (Millipore, Bedford, MA, USA) to remove large EVs, followed by ultracentrifugation twice at 100,000 × g for 114 minutes using an SW 32 Ti Rotor Swinging Bucket rotor (K factor of 256.8, 28,536 r/min; Beckman Coulter, Fullerton, CA, USA) to pellet the iMSC-sEVs. Finally, the pellet was used for identification experiments or resuspended in phosphate-buffered saline (PBS) and stored at –80°C. All centrifugation steps were performed at 4°C.

iMSC-sEVs were extracted by centrifugation protocols as previously described31 (link) from the conditioned medium of iMSC. Briefly, the conditioned medium was collected and centrifuged at 300g for 10 min and 2000g for 20 min to remove cells, dead cells, respectively. Afterward, the supernatant was centrifuged at 10,000g for 30 min and filtered through a 0.22-μm filter sterilize Steritop™ (Millipore) to remove cellular debris and microvesicles. This collected supernatant was subsequently subjected to ultracentrifugation at 100,000 × g for 70 min in an SW 32 Ti Rotor Swinging Bucket rotor (k factor of 256.8, 28536 rpm, Beckman Coulter, Fullerton, CA) to pellet sEVs. After removing the supernatant, the pellet was resuspended in phosphate buffer saline (PBS), followed by another ultracentrifugation at 100,000g for 70 min. Finally, pelleted sEVs were resuspended in PBS stored at −80°C. All steps were performed at 4°C under sterile conditions.