Isolated Exos were confirmed via flow cytometry analysis with CellTrace™ Violet (ThermoFisher Scientific, Waltham, MA, USA) as previously published [26 (link)]. In brief, isolated Exos were incubated with CellTrace reagent for 20 min at 37 °C. Following labeling, flow cytometry analysis was accomplished using the CytExpert Software v2.5 for the CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA).
Exo pellet was then characterized through a western blot for specific protein biomarkers for exosomes, namely CD63 and heat shock protein 70 (HSP70), as published [9 (link)]. In brief, exosomal proteins were extracted by reconstitution with radioimmunoprecipitation assay lysis buffer (RIPA), and the concentration of isolated protein was acquired using the Bio-Rad protein assay. The extracted protein (50 μg) and ladder (SeeBlue Plus2 Prestained Standard) [38 (link)] were loaded onto 4–12% Bolt gels and ran for 22 min at 200 V, followed by transfer to a polyvinylidene difluoride (PVDF) membrane using the iBlot2 Gel Transfer Device. Membranes were then incubated with antibodies CD63 (1:1000; SBI, Palo Alto, CA, USA) and HSP70 (1:1000; SBI, Palo Alto, CA, USA).
Exo pellet was then characterized through a western blot for specific protein biomarkers for exosomes, namely CD63 and heat shock protein 70 (HSP70), as published [9 (link)]. In brief, exosomal proteins were extracted by reconstitution with radioimmunoprecipitation assay lysis buffer (RIPA), and the concentration of isolated protein was acquired using the Bio-Rad protein assay. The extracted protein (50 μg) and ladder (SeeBlue Plus2 Prestained Standard) [38 (link)] were loaded onto 4–12% Bolt gels and ran for 22 min at 200 V, followed by transfer to a polyvinylidene difluoride (PVDF) membrane using the iBlot2 Gel Transfer Device. Membranes were then incubated with antibodies CD63 (1:1000; SBI, Palo Alto, CA, USA) and HSP70 (1:1000; SBI, Palo Alto, CA, USA).
Free full text: Click here
