Fig 1: Characterization of the HDL removal from the fractionated EV and yield/quality of the isolated EV.a Schematic of the EV fractionation and immunocapture of HDL. The diluted plasma (sample I) runs through the asymmetric nanoporous membrane to remove other particles with a size-exclusion mechanism, thus obtaining the EV fraction (sample II). Due to the dominating amount of HDL, a small portion of HDL remained in the EV fraction, so it was incubated with anti-ApoA1 and anti-ApoB antibodies, and then magnetic nanobeads conjugated with anti-rabbit IgG antibodies and run through the MNM device to remove the HDL, and the flow-through was collected (sample III). b Lipoproteins (cholesterol) remnant at different stages of purification from samples I, II, III in a), and (inset) EV concentration before and after MNM immunocapture (sample II and III) (n = 5 measurements for each sample). c Size distribution of EV samples with NTA before (sample II) and after MNM immunocapture (sample III) showing minimal EV loss or lysis/coalescence. (inset) CD63 and CD9 concentration before and after MNM immunocapture (sample II and III) measured by ELISA (n = 2 and 3 measurements for CD63, CD9, respectively). Error bars indicate the standard deviation (SD) in each plot.
Fig 2: Characterization of MNM immunocapture of specific EV, i.e., EV with EGFR.a Schematic of the EV fractionation and immunocapture of EV with EGFR. The DiFi cell culture medium was run through the asymmetric nanoporous membrane to obtain the EV fraction by size separation (sample I), which was then incubated with the anti-EGFR antibodies and then magnetic nanobeads conjugated with anti-human IgG antibodies. Then the samples passed through the MNM device, with the captured sample on the magnetic membrane mixed with lysing buffer (sample II), and the flow-through was collected (sample III). b Hm-miR-21 expression level of different DiFi exosome fractions from samples I, II, III in (a) (n = 3 measurements for each sample). (inlet) The total amount of EGFR was measured for I and III, indicating most EGFR-positive EVs from the ANM-isolated EVs are captured by MNM (n = 7 measurements for each sample). c CD63 and CD9 concentration of ANM isolate (sample I) and MNM EGFR isolate (sample II) measured by ELISA. (n = 3 measurements for each sample) d Expression level of Hm-miR-21 in the EGFR exosomes before and after 8 × dilution of the DiFi samples and the Ct value of their qRT-PCR results (n = 5 measurements for each sample). e Western blot of the isolated EVs from the DiFi cell line. After the Marker Lane then (1) ANM-isolated sEV; (2) MNM bead captured sEV. Each well was loaded with 40 µg protein. Odyssey exposure (bottom: 10 min, top: overexposed). The top panel is blotted by EGFR and the bottom by syntenin-1, respectively. Error bars indicate the standard deviation (SD) in each plot.
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