Ez c1 bronze version 3

The cellular uptake of Au NCs in cells was assessed using the Nikon Eclipse Te2000-U microscope (Nikon, Yokohama, Japan) with the confocal laser scanning system C1si (capable of 32-bit spectral imaging). Imaging was performed using a 60×/1.4 NA oil immersion objective (Plan Apo VC, Nikon, Yokohama, Japan). The BSA-Au NCs, BSA-Alexa, and propidium iodide were excited at 488 nm with argon-ion laser and Au-MES NCs and nucleus stain Hoechst 33258 were excited at 404 nm with diode laser.
For investigation of uptake mechanisms of BSA-Au NCs, co-localization with endocytosis markers has been studied. GFP in transfected endosomes and lysosomes were excited at 488 nm with argon-ion laser and BSA-Au NCs were excited at 543 nm. Co-localization of BSA-Au NCs and GFP in superimposed images appear yellow.
The three-channel RGB detector (band-pass filters 450/17, 545/45 and 688/67 for blue, green, and red channels, respectively) was used. The cells were maintained at 37 °C in Microscope Stage Incubation System (OkoLab, Pozzuoli, Italy) in a humidified atmosphere containing 5% of CO₂ (0.80 Nl/min O₂ and 0.04 Nl/min CO₂). Image processing was performed using the Nikon EZ-C1 Bronze version 3.80 and ImageJ 1.46 software (free non-commercial software developed at the National Institutes of Health, Bethesda, MD, USA).

For intracellular imaging studies, MCF-7 and MDA-MB-231 cells were seeded into an 8-chambered cover glass plate (Thermo Fisher, USA) with a density of 3·10⁴ cells/chamber. For the evaluation of Au NCs uptake and intracellular localization, cells were treated with 13.75 mg/mL of Au NCs and incubated for the next 24 h. Nuclei of the cells were stained with 0.01 mg/mL Hoechst 33258 (Sigma-Aldrich, Steinheim am Albuch, Baden-Württemberg, Germany).
The accumulation of Au NCs was observed using a Nikon Eclipse Te2000-S C1 Plus laser scanning confocal microscope (Nikon, Minato-ku, Japan) equipped with a diode laser for 404 nm wavelength excitation and an argon laser for 488 nm wavelength excitation. Imaging was performed using 60×/1.4 NA oil immersion objective (Nikon, Japan). The three-channel RGB detector filters (band-pass filters 450/17, 545/45 and 688/67 for blue, green, and red channels, respectively) were used. Hoechst 33258 was excited at 404 nm, Au NCs was excited at 488 nm. The cells were incubated at 37 °C in the Microscope Stage Incubation System (OkoLab, Pozzuoli, Italy) in a humidified atmosphere containing 5% of CO₂ (0.80 Nl/min O₂ and 0.04 Nl/min CO₂) during imaging. Image processing was performed using the Nikon EZ-C1 Bronze version 3.80 and ImageJ 1.46 software.