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. 2020 Dec 17;11:576516. doi: 10.3389/fimmu.2020.576516

Figure 6.

Figure 6

Patients with unstable CAD have increased uEV miR-155 expression and alterations in CD45+, CD11b+, and CD16+ uEVs. For qRT-PCR analysis, four EV pellets (derived from 6-ml urine) were re-suspended in TRIzol™ for RNA extraction. 100ng of RNA was reverse transcribed to cDNA and qRT-PCR analysis was performed for (A) miR-155 (n = 11 per group) and (B) miR-21 (5 stable and 9 unstable CAD patients) expression using the reference genes, RNU6B and RNU48. For flow cytometry analysis, four pellets were re-suspended in 300 μl of sterile-filtered PBS, 50 µl of each sample was stained with the membrane stain, Bodipy and the antibodies CD45, CD11b, CD14, and CD16 (n = 1 per group). Ten thousand EVs positive for Bodipy were analyzed. (C) The percentage of CD45 and CD11b fluorescence antibody relative to Bodipy stain was analyzed. (D) The concentration of CD45 and CD11b fluorescent EVs per ml was analyzed. (E) The percentage of CD14 and CD16 fluorescence antibody relative to Bodipy stain was analyzed. (F) The concentration of CD14 and CD16 fluorescent EVs per ml was analyzed. Statistical analysis was performed using non-parametric Mann-Whitney U tests where *p < 0.05, **p < 0.01, and ***p < 0.001 represent statistical significance. Significance between groups is indicated by capped lines.