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. 2021 Nov 26;10(12):3321. doi: 10.3390/cells10123321

Figure 2.

Figure 2

AML-EV purification process using sequential TFF. AML cell lines were cultured in a particle-poor RPMI medium using ITS+1 as serum replacement. (A) Particle concentration was measured with tunable resistive pulse sensing (TRPS) using a 150 nm pore (green bars). Protein amount was measured using a detergent compatible (DC) protein assay (red bars). Limit of detection (LOD) of the TRPS measurement using the 150 nm pore is depicted as a green dotted line. The red dotted line represents the LOD of the DC protein assay. Fractions analyzed as indicated (conditioned medium, CM; soluble factors, solF, cycles 1 and 2; tangential flow filtration EV-containing retentate, TFF, cycles 1 and 2). (B) Recovery compared to total amount of input particles showing mean 89.97% recovery after TFF1 purification and mean 66.23% recovery of total particles after TFF2. The recovery of particles within the solF fractions was less than 5% for solF1 and solF2 suggesting a loss of EVs in the column during TFF2. (C) Representative Western blot analysis of identical sample volumes (20 µL) of EV purification fractions of MOLM-14 EVs. (D) Quantification by densitometry showing significant enrichment of EV-markers CD9, CD63, CD81 and flotillin-1 by TFF. (A,B), n = 4, one-way ANOVA, ** p < 0.01; (C,D), n = 2).