Objective: To confirm the presence of known EV protein and RNA markers in EXO-NET isolated EVs from human plasma.

Method: Pooled normal human plasma (500µl) was incubated with 30 µl of EXO-NET for 15 min. EXO-NET-captured EVs were magnetically isolated and lysed for downstream analysis. Total EV protein and RNA were extracted and analysed by Western blot and qPCR.

Result: Western blot analysis showed EXO-NET-isolated EVs from normal human plasma were positive for CD63, CD81, CD9, TSG101 and Flotillin-1, and negative for calnexin (an EV negative marker). qPCR analysis demonstrated that EXO-NET isolated EVs had high yield of mRNA (GAPDH) and microRNA (miR-191).

Objective: To confirm the presence of known EV protein and RNA markers in EXO-NET isolated EVs from human saliva.

Method: Pooled normal human saliva (400µl) was diluted with 400µl of PBS and then incubated with 30 µl  of EXO-NET for 15 min.  EXO-NET captured EVs were magnetically isolated and lysed for downstream analysis.  Total protein and RNA were extracted from captured EVs and analysed by Western blot and qPCR.

Result: Western blot analysis confirmed the presences of CD63, CD81 and CD9. Calnexin (EVs negative marker) was not detectable. qPCR analysis confirmed that EXO-NET isolated EVs had high yield of mRNA (GAPDH) and microRNA (U6).

Objective: To confirm the presence of known EV protein and RNA markers in EXO-NET isolated EVs from MCF-7 cell conditioned medium.

Method: MCF-7 cells were cultured in growth medium (MEM) + 10% FBS + 1X GlutaMAXTM Supplement) to ~80% confluency. Cells were incubated in serum free Essential 8 (E8) media or basal MEM media supplemented with 1X GlutaMAXTM (MEG) for 24 h.  Cell-conditioned medium (CCM, 20mL) was incubated with 30 µl of EXO-NET for 30 min.  EXO-NET captured EVs were magnetically separated from the CCM.

Result: Western blot analysis confirmed presence of CD9 and Flotillin-1.  qPCR demonstrated that EXO-NET isolated EVs had higher yield of mRNA (GAPDH) and microRNA (U6 and miR191) in E8-CCM than MEG-CCM.