MSC-Exo were labeled with the C5 Maleimide-Alexa 594 (CM-A954; Invitrogen, A10256, California, USA) as previously described 31 (link). CM-A954 (200 μg/ml - 2.5 µl) was added to 50μl MSC-Exo (1.5 mg/ml) for 60 min in the dark (25°C), exosome spin columns (MW3000, Invitrogen) were prepared according to manufacturer's instructions, and powdered resin was hydrated for 25 min at room temperature. Spin columns in the collection tubes were centrifuged (750g) for 2 min using AllegraX-15R (Beckman, USA), and the columns were placed in 1.5 ml tubes and centrifuged for 3 min (750g) to collect labeled MSC-Exo. Non-incorporated or excess dye was retained by the resin and controls involving CM-A954, but no MSC-Exo were performed in parallel to confirm dye retention by the column. To avoid dye-stained aggregates, the labeled MSC-Exo were filtered through a 0.22 μm syringe filter immediately before use.
Xian P., Hei Y., Wang R., Wang T., Yang J., Li J., Di Z., Liu Z., Baskys A., Liu W., Wu S, & Long Q. (2019). Mesenchymal stem cell-derived exosomes as a nanotherapeutic agent for amelioration of inflammation-induced astrocyte alterations in mice. Theranostics, 9(20), 5956-5975.
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