Breast cancer cells were imaged using a confocal Nikon Eclipse Te2000-U microscope (Nikon, Yokohama, Japan) with a C1si laser scanning confocal system. The microscope was equipped with a Fianium WhiteLase Micro supercontinuum laser (NKT Photonics, Birkerød, Denmark) with a pulse repetition rate of 30 MHz and filtered using 410 ± 5 nm (for Hoechst), 480 ± 5 nm (for BDP-H and BDP-NO2) and 560 ± 5 nm (for all red-emitting commercial probes) band-pass filters (all from Thorlabs Inc., Newton, NJ, USA). Imaging was performed using an 60×/1.4 NA oil immersion objective (Plan Apo VC, Nikon, Yokohama, Japan). The 32-channel spectral detector was applied to investigate the fluorescence of BDP-NO2 and BDP-H in live cells. The brightfield mode of Nikon Eclipse Te2000-U and the three-channel RGB detector with band-pass filters of 450/34 nm, 546/89 nm and 688/134 nm for blue, green and red channels, respectively, were used. Live cells were maintained 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). Image processing was performed using the EZ-C1 Bronze software (v.3.80, Nikon, Tokyo, Japan) and ImageJ software (v.1.53e, U.S. National Institutes of Health, Bethesda, MD, USA) [65 (link)].
Microscope stage incubation system
Manufactured by Okolab
Sourced in Italy
The Microscope Stage Incubation System is a laboratory equipment designed to maintain stable environmental conditions around a microscope stage. It provides control over temperature, humidity, and gas composition to create an optimal environment for cell and tissue samples during long-term microscopy observations.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse te2000 u microscope, by Nikon (2 mentions)
Plan apo vc, by Nikon (2 mentions)
Ez c1 bronze version 3, by Nikon (2 mentions)
8 chambered cover glass plate, by Thermo Fisher Scientific (2 mentions)
Hoechst 33258, by Merck Group (2 mentions)
Fianium whitelase micro supercontinuum laser, by NKT Photonics (1 mentions)
Eclipse te2000 u, by Nikon (1 mentions)
1.4 na oil immersion objective, by Nikon (1 mentions)
Fls920, by Edinburgh Instruments (1 mentions)
4 protocols using microscope stage incubation system
1
Confocal Imaging of Breast Cancer Cells
2
Cellular Uptake of Au Nanocrystals
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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 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).
3
Intracellular Imaging of Au NCs Uptake
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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.
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.
4
Intracellular Imaging and Quantification of 99mTc-BSA-Au NCs
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For intracellular imaging studies, HEK-293T cells were seeded into an 8-chambered cover glass plate (ThermoFisher, Rochaster, NY, USA) with a density of 3 × 104 cells/chamber. For the evaluation of 99mTc-BSA-Au NCs uptake and intracellular localization, cells were treated with 15 mg/mL of 99mTc-BSA-Au NCs and incubated for different periods of time. Nuclei of the cells were stained with 0.01 mg/mL Hoechst 33258 (Sigma-Aldrich, Germany). The accumulation of 99mTc-BSA-Au NCs was observed using confocal laser scanning microscopy, as describe before. Hoechst 33258 was excited at 404 nm, 99mTc-BSA-Au NCs was excited at 488 nm. During imaging 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).
Additionally, accumulation dynamics of 99mTc-BSA-Au NCs in HEK-293T cells was spectroscopically evaluated using Edinburgh spectrometer FLS920, measuring emission intensity of cell suspensions. Accumulation dynamics is determined as integral of emission intensity (600–700 nm) at the specific time divided by cell number at that time in the suspension and plotted as emission intensity per cell. Experiments with cells were performed in triplicate and repeated three times. Data are expressed as mean ± standard deviation (SD).
Additionally, accumulation dynamics of 99mTc-BSA-Au NCs in HEK-293T cells was spectroscopically evaluated using Edinburgh spectrometer FLS920, measuring emission intensity of cell suspensions. Accumulation dynamics is determined as integral of emission intensity (600–700 nm) at the specific time divided by cell number at that time in the suspension and plotted as emission intensity per cell. Experiments with cells were performed in triplicate and repeated three times. Data are expressed as mean ± standard deviation (SD).
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