Exoquick tc exosome isolation reagent

CPC-exosome isolation was performed using ExoQuick-TC™ Exosome Isolation reagent (System Biosciences, Palo Alto, CA, USA), following the procedure outlined by the manufacturer. To prepare the exosome media prior to ExoQuick treatment, the concentration was increased from 50 to 130 µl using an Amicon Ultra filter (EMD Millipore, Billerica, MA, USA) with a 100,000-molecular weight cutoff (14 (link)).

HucMSCs were provided by Shandong Qilu cell therapy Engineering Technology Co., Ltd. For hucMSC-exo extraction, hucMSCs at 3–5 passages were used according to our previously described methods.19 (link) Briefly, hucMSCs culture supernatant was collected and centrifuged at 3000 g for 15 min to remove dead cells and cell debris. The resultant supernatant was filtered once through 0.22 µM pore filter (Merck KGaA, Darmstadt, Germany) to eliminate cell debris and bacteria. The filtered supernatant was concentrated with a 150-kD Protein Concentrator (Millipore, Massachusetts, USA) and filtered once again using 0.22-μM pore filter to eliminate bacteria further. The final supernatant was subject to ExoQuick-TC exosome isolation reagent (5: 1, v/v; System Biosciences, California, USA) and incubated at 4°C for 12 h. Then, the exosomes pellet was collected after centrifuging at 15,000 g for 30 min and resuspended in PBS. Finally, it was stored at −80°C after quantified the protein concentration by the BCA method.
For characterization, hucMSC-exo were detected using transmission electron microscopy (TEM) to observe the morphology and size. Particle size distribution was assessed by flow nano-Analyser (NanoFCM, Xiamen, China). Finally, the typical exosomal markers were validated by Western blot. Detailed procedures are provided in Supplementary Materials.