Lsm 880 meta confocal laser scanning microscope

hMSCs (3 × 10⁵ cells/well) were seeded in 6-well plates and cultured at 37 °C in a 5% CO₂ atmosphere, after which they were rinsed twice and pre-incubated for 1 h with 2 ml of Opti-MEM medium at 37 °C. pDNA-coupled SF-NPs were added to the cells and incubated for 6 h at 37 °C. The hMSCs were then washed three times with 1 ml of PBS to remove any free gene complexes, suspended in PBS, and incubated for 24 h. The cells were detached to obtain SF-NP-treated T1 cells, and the remaining T1 cells were subcultured three times to obtain T4 cells. To determine transfection efficiency, the cells were harvested and analyzed using a flow cytometer (Guava Technologies, Hayward, CA, USA) equipped with a 488/642-nm excitation laser. Data shown are the mean fluorescent signals for 10,000 cells.
For confocal microscopy, the cells were fixed with 4% paraformaldehyde, mounted in Fluorescence Mounting Medium (Dako Cytomation), and visualized on an LSM 880 Meta confocal laser-scanning microscope (Zeiss). Fluorescence was monitored in appropriate channels to detect DAPI (4′,6-diamidino-2-phenylindole; excitation, 358 nm; emission, 461 nm), EGFP (excitation, 488 nm; emission, 530 nm), and SF-NPs (excitation, 610 nm; emission, 655 nm).

hMSCs (3 × 10⁵ cells/well) were seeded in 6-well plates and cultured at 37 °C in a 5% CO₂ atmosphere, after which they were rinsed twice and pre-incubated for 0.5 h at 37 °C in 2 ml of Opti-MEM medium. pDNA-coupled SF-NPs were added to the cells and incubated for 30 min to 6 h. Thereafter, the cells were detached to obtain SF-NP-treated T1 cells, and the remaining T1 cells were subcultured three times to obtain T4 cells.
For TEM, cells were collected by centrifugation at 850 g and fixed with 2.5% glutaraldehyde. The fixed cells were subjected to dehydration using a graded ethanol series (20%, 40%, 60%, 80%, and 100%), and then embedded in Epon 812 resin. The resin blocks were sectioned using a microtome and imaged on a JEOL 200CX TEM (80 kV) microscope equipped with an AMT (Advanced Microscopy Techniques) camera system.
For confocal microscopy, the cells were fixed with 4% paraformaldehyde, mounted in Fluorescence Mounting Medium (Dako Cytomation, Hamburg, Germany), and visualized on an LSM 880 Meta confocal laser-scanning microscope (Zeiss, Oberkochen, Germany). Fluorescence was monitored in appropriate channels to detect CellLight® Early Endosomes-GFP (excitation, 485 nm; emission, 520 nm) and SF-NPs (excitation, 610 nm; emission, 655 nm).