The uptake of FITC-labeled dPG-NGs with and without CC in A549, Hela, and MCF7 cells was analyzed using confocal laser scanning microscopy (CLSM). The cells were propagated as described above. For CLSM, HeLa cells were seeded in eight-well ibidi slides (ibidi treat) in 270 µL of DMEM. After cell attachment for 4 h to 24 h, 30 µL of a post-seeding solution containing compound was added, and the cells were further incubated overnight. Before imaging, the cells were stained with Hoechst 33342 (1 µg·mL−1), washed twice with PBS, and covered with fresh cell culture medium (DMEM). Confocal images were taken with an inverted confocal laser scanning microscope Leica DMI6000CSB SP8 (Leica, Wetzlar, Germany) with a 63×/1.4 HC PL APO CS2 oil immersion objective using the manufacturer-given LAS X software in sequential mode with the following channel settings: transmission Ch (gray intensity values), excitation laser line 405 nm, detection of transmitted light (photomultiplier); Ch1 (Hoechst 33342): excitation laser line 405 nm, detection range 410 nm–484 nm (hybrid detector); Ch2 (FITC): excitation laser line 488 nm, detection range 493 nm–712 nm (hybrid detector).
Dmi6000csb sp8
Manufactured by Leica
Sourced in Germany
The Leica DMI6000CSB SP8 is a research-grade inverted microscope designed for advanced imaging applications. It features a motorized stage, condensers, and objectives, enabling precise control and automation of the imaging process. The microscope is equipped with a SP8 laser scanning confocal system, providing high-resolution, multicolor imaging capabilities.
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4 protocols using dmi6000csb sp8
1
Cellular Uptake of FITC-labeled dPG-NGs
2
Confocal Microscopy of Cancer Cell Lines
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For confocal laser scanning microscopy, the epithelial human cancer cell line A431 (DSMZ No. ACC 91) and the human lung carcinoma epithelial cell line A549 (DSMZ No. ACC 107) were routinely propagated in Dulbecco's modified Eagle's medium (DMEM), with 2 % glutamine, penicillin/streptomycin (all from Gibco BRL, Eggenstein, Germany), and 10 % fetal calf serum (FCS, Biochrom AG, Berlin, Germany) at 37 °C with 5 % CO2 and were subcultured twice a week. For confocal microscopy, 27 000 cells were seeded in each well of a μ‐Slides 8 Well (ibidi GmbH, Martinsried, Germany) and were cultured at 37 °C for 24 h. Thereafter, dye‐labeled test substances were added to the cells at a final concentration of 1 μm. Confocal images were taken with an inverted confocal laser scanning microscope Leica DMI6000CSB SP8 (Leica, Wetzlar, Germany) with a 63×/1.4 HC PL APO CS2 oil immersion objective at 37 °C using the manufacture given LAS X software. Images of different groups were acquired with the same laser and detector settings by using the Leica LAS AF software. The fluorescence detection was performed sequentially for each channel set with the acousto optical beam splitter between λ=570 and 648 nm for the ICC dye and between λ=650 and 749 nm for IDCC dyes. ICC was excited using the λ=561 nm diode‐pumped solid‐state laser line, whereas IDCC dyes were excited with a λ=633 nm HeNe laser.
3
Confocal Imaging of Cy5-Labeled DR-dPGS and DR-dPGS@DOX in HeLa Cells
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The uptake of Cy5-labeled DR-dPGS and DR-dPGS@DOX in HeLa cells was analyzed using Confocal Laser Scanning Microscopy (CLSM). The cells were propagated as described above. For CLSM, HeLa cells were seeded in 8-well ibidi slides (ibidi treat) in 270 µL DMEM. After cell attachment 4 h to 24 h, a postseeding 30 µL of solution containing compound were added for 3 h or 20 h. Before imaging, the cells were stained with Hoechst 33342 (1 µg/mL), washed with PBS and covered with fresh cell culture medium (DMEM). Confocal images were taken with an inverted confocal laser scanning microscope Leica DMI6000CSB SP8 (Leica, Wetzlar, Germany) with a 63x/1.4 HC PL APO CS2 oil immersion objective using the manufacture given LAS X software in sequential mode with the following channel settings: Transmission Ch (grey intensity values), excitation laser line 405 nm, detection of transmitted light (photomultiplier); Ch1 (Hoechst 33342): excitation laser line 405 nm, detection range 410 nm–485 nm (hybrid detector); Ch2 (Doxorubicin): excitation laser line 488 nm, detection range 496 nm–629 nm (hybrid detector); Ch3 (Cy5 dye): excitation laser line 488 nm, detection range 638 nm–797 nm (photomultiplier).
4
Cellular Uptake of Labeled Glycosaminoglycans
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The uptake of IDCC-labeled hPGN-Gs and hPGS-Gs by HaCat cells was analyzed using confocal laser scanning microscopy (CLSM). The cells were propagated as described above. For CLSM, HaCat cells were seeded in eight-well ibidi slides (ibidi treat) in 270 μL of DMEM. After cell attachment for 4 h to 24 h, 30 μL of a postseeding solution containing compound was added, and the cells were further incubated overnight. Before imaging, the cells were stained with Hoechst 33342 (1 μg mL−1), washed twice with PBS, and covered with fresh cell culture medium (DMEM). Confocal images were taken with an inverted confocal laser scanning microscope Leica DMI6000CSB SP8 (Leica, Wetzlar, Germany) with a 63×/1.4 HC PL APO CS2 oil immersion objective using the manufacturer-given LAS X software in sequential mode with the following channel settings: transmission Ch (gray intensity values), excitation laser line 405 nm, detection of transmitted light (photomultiplier); Ch1 (Hoechst 33342): excitation laser line 405 nm, detection range 410–484 nm (hybrid detector); Ch2: excitation laser line 488 nm, detection range 493–712 nm (hybrid detector).
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