miRNAs regulate MDDC differentiation. Monocytes were untransfected, transfected with 100 nM Cy3-labeled nontargeting negative anti-miR, or transfected with a 1:5 ratio of Cy3-labeled nontargeting negative anti-miR and anti–miR-21, anti–miR-34a, anti–let-7e, anti–miR-99b, anti–miR-125a, or anti-miR-342 (100 nM). (A) FACS analysis showing (from left to right): live cell gating (R1), percentage of Cy3 positive cells (R2) (positive cells gated based on live cell gates), and cell surface phenotype (R1 + R2) during MDDC differentiation at day 5 with the indicated miRNA inhibitors. Numbers in each quadrant represent percentage of positive cells. Data from 1 representative donor (of 3) are shown. (B) DC-SIGN/CD14 expression was normalized to MFI ratios of negative anti-miR transfected MDDCs at day 5. Ratios were calculated using MFI of DC-SIGN/MFI of CD14. P values were calculated using a paired t test comparison with the negative anti-miR transfected MDDCs: *P < .05; **P < .01; ***P < .001. Scatter plots show data from 3 independent donors. Monocytes were differentiated in the presence of GM-CSF and IL-4. Cells were stained and analyzed at day 5 of MDDC differentiation for DC-SIGN and CD14 cell surface expression. Live cell populations were further gated on the positively transfected (Cy3⁺) cells for analysis.