Ix71 stand

Nuclear uptake into HeLa cells was studied via wide-field fluorescence microscopy. Cultured cells were seeded in eight-well glass bottom µ-slides (ibidi) using DMEM with 10% (vol/vol) FBS as nutrition medium and grown until they reached a confluency of 50 to 80%. In all live-cell studies, we stained the cell nuclei with Hoechst (Thermo Fisher) and used DMEM without phenol red (Gibco). To begin with the experimental assay, a concentration of 0.6 nM Bodipy 630/650-labeled NLS-NCs or 0.6 nM Nile Red 552/636-labeled blank NCs were added to the cell medium. The cells were then transferred to an Olympus IX71 stand that was preheated to 37 °C with a 5% CO₂ atmosphere. A DeltaVision core wide-field fluorescence microscope was equipped with a Photometrics CoolSNAP HQ² camera coupled to an interline CCD transfer and was operated via SoftWorx 4.12 software. A 60× oil objective was applied for imaging. Relative nuclear fluorescence intensities were determined via signal colocalization with the chromatin stain Hoechst. NC uptake kinetics was followed over 12 h with time-lapse images taken every 30 min for the first 3 h, followed by images being recorded every 1 h for the next 9 h. Cell studies were repeated three times at each experimental condition.

High-resolution imaging was performed using a General Electric (Boston, MA) DeltaVision Elite imaging system mounted on an Olympus (Japan) IX71 stand with a motorized XYZ stage, Ultimate Focus, cage incubator, ultrafast solid-state illumination with excitation/emission filter sets for DAPI, CFP, FITC, GFP, YFP, TRITC, mCherry, and Cy5, critical illumination, Olympus PlanApo N 60X/1.42 NA DIC (oil) and UPlanSApo 60X/1.3 NA DIC (silicone) objectives, Photometrics (Tucson, AZ) CoolSNAP HQ2 camera, SoftWoRx software with constrained iterative deconvolution, and vibration isolation table.