2.8. Fluorescent Imaging Analyses of the Pre-miRNA Transfected HeLa Cells

The HeLa cells (70,000 cells/mL) were split on the bottom of a 35 mm dish in a 2 mL medium and cultured overnight. For the lipofection, the transfections of the HeLa cells with the modified pre-miR17 (final concentrations = 30 nM) were performed with Lipofectamine^TM 2000 (Invitrogen) in 96-well plates according to the manufacturer’s instructions and incubated for 12 h. For the methods of loading the beads, Opti-MEM containing the modified pre-miR17 (2 μM) was added to the dish after removing the medium, and the cells were incubated for 2 min. The glass beads (2 mg, <106 µm, acid washed, Sigma Aldrich, St. Louis, MO, USA) were added to the medium, after which the dish was shaken for 30 s. The HeLa cells were incubated for 1 h at 37 °C. The cells were washed with Opti-MEM, and the medium was changed to DMEM without phenol red.

For immunofluorescent analyses, the HeLa cells were fixed in phosphate-buffered saline (PBS) containing 4% paraformaldehyde and treated with PBS containing 0.2% Triton-X100 for 5 min at RT. Subsequently, the cells were stained with anti-AGO2 (FUJIFILM Wako, Osaka, Japan) and Alexa Fluor 648-conjugated anti-mouse IgG antibodies (Abcam, Cambridge, UK), anti-GW182 (Santa Cruz Biotechnology, Dallas, TX, USA) and Alexa Fluor 488-conjugated anti-goat IgG antibodies (Invitrogen, Carlsbad, CA, USA), or anti-Dicer (Abcam) and Alexa Fluor 647-conjugated anti-mouse IgG antibodies (Abcam). For time course analysis, after introduction of the miStem into the cell by use of the beads loading method, images of the cells were obtained at specific time point. The HeLa cells were visualized on an FV-1000 or FV-3000 confocal laser microscopy (Olympus, Tokyo, Japan). The fluorescent signals were recorded as follows: Cy3: Ex = 543 nm, Em = 555–625 nm; Alexa647: Ex = 633 nm, Em 645–745 nm; FRET: Ex = 458 nm, Em = 555–625 nm. The fluorescent images were processed with ImageJ.