Lsm 510 meta confocal microscpe

An Olympus IX-71 inverted fluorescence microscope equipped with a true-color charge-coupled device (QColor5, Olympus), a LSM 510 Meta confocal microscpe (Zeiss, Dublin, CA) and a hyper-spectral imaging camera (Nuance, 420–720 nm spectral range, CRI, now Advanced Molecular Vision) were used for cell imaging. Low-magnification images were obtained with a 20X objective (NA 0.75, Olympus) and high-magnification with 40X and 100X oil-immersion objectives (NA 1.40, Olympus). Wide UV filter cube (330–385 nm band-pass excitation, 420 nm long-pass emission, Olympus) was used for imaging of all QD probes. All images were acquired with cells attached to the coverslip bottom of the well and immersed in PBS without anti-fading reagents. For quantitative comparisons, the same exposure time and gain were applied during imaging. Nuance image analysis software and ImageJ were used to identify regions of interest that included stained cells and excluded ‘blank’ cell-free areas. Average fluorescence intensity throughout all regions of interest within a single image was recorded. Identical analysis was performed on 4 images (containing ~40 cells per field of view) taken from different areas of the sample to obtain an overall average staining intensity and assess signal variation.

To evaluate the cell binding ability of RNP₈, it was incubated with LNCaP, 22RV1 and PC-3 cells at RT to minimize internalization. Briefly, RNP₈ (the siRNA in it was fluorescently labeled with Alexa Fluor 555) and cells (~40,000) were incubated for 1 h in RPMI 1640 supplemented with 1% BSA. The cells were washed with PBS and their fluorescence was measured on a LSR II flow cytometer (Beckton Dickinson, Franklin Lakes, NJ). A representative gating strategy for the flow cytometry analysis is shown in Supplementary Fig. 19. For cellular uptake, RNP₈ incubation with cells was performed at 37°C. At the end of the incubation, cells were lysed with PBS containing 0.25% Triton X-100, and the uptake was measured by quantifying the siRNA in RNP₈ in cell lysate using stem-loop real-time PCR^{53 (link)}. To image RNP₈ intracellular trafficking, dual-color fluorescence-labeled RNP₈ (siRNA labeled with Alexa Fluor 555 and the dsRBD octamer labeled with Alexa Fluor 488) was incubated with LNCaP cells at 37°C and imaged at 0 min, 15 min, 1 h, 3 h and 6 h. In a separate experiment, single-color fluorescence-labeled RNP₈ (only the siRNA is labeled with Alexa Fluor 555) was incubated with cells together with LysoTracker Green DND-26 (Thermofisher, Waltham, MA). After counter-stained with Hoechst, the cells were analyzed with a LSM 510 Meta confocal microscpe (Zeiss, Dublin, CA).