Transfection of fluorescent-protein-tagged plasmids or siRNAs into A172 cells was carried out as described above. After electroporation, cells were seeded on μ-Dish 35 mm high (IBIDI) and incubated at 37°C for 24-36h. Cells were then washed with PBS, irradiated with UV (40 J/m2). Microscopic analysis was done in normal medium containing 1 μg/ml Hoechst 33342 (Life technologies) for nuclear staining. Microscopic images were obtained by using a Leica TCS SP5 DMI6000 CS Confocal Microscope (Leica), equipped with ultraviolet 405 Diode, Argon, DPS3561, HeNe594 Lasers. Fluorescent images were captured with a 63.0 × lens zoomed in 1-3× with a 1,024×1,024 frame and 400 Hz scanning speed. For multicolor experiments, the following wavelength settings were used; mCherry (Ex 594 nm/Em 604-766 nm), EGFP (Ex 488 nm/Em 498-567 nm), YFP (Ex 514 nm/Em 520-590 nm), dsRed (Ex 561 nm/Em 571 -723 nm), and Hoechst (Ex 405 m/Em 414-475 nm). Images were further analyzed using Leica LAS AF software (Leica) and Image J (NIH).
Tcs sp5 dmi6000 cs confocal microscope
Manufactured by Leica
The Leica TCS SP5 DMI6000 CS Confocal Microscope is a high-performance research-grade instrument designed for advanced imaging and analysis. It features a spectral detection system and hybrid detectors for optimal signal-to-noise ratio and resolution. The microscope is equipped with a motorized stage and various laser lines to enable versatile imaging capabilities.
Automatically generated - may contain errors
Lab products found in correlation
μ dish 35 mm high, by Ibidi (2 mentions)
Las af software, by Leica (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Bacmam 2, by Thermo Fisher Scientific (1 mentions)
Las x software, by Leica (1 mentions)
Sir dna reagent, by Cytoskeleton (1 mentions)
Celllight tubulin gfp, by Thermo Fisher Scientific (1 mentions)
3 protocols using tcs sp5 dmi6000 cs confocal microscope
1
Fluorescent Microscopy Imaging of Transfected A172 Cells
2
Fluorescent Microscopy Imaging of Transfected A172 Cells
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Transfection of fluorescent-protein-tagged plasmids or siRNAs into A172 cells was carried out as described above. After electroporation, cells were seeded on μ-Dish 35 mm high (IBIDI) and incubated at 37°C for 24-36h. Cells were then washed with PBS, irradiated with UV (40 J/m2). Microscopic analysis was done in normal medium containing 1 μg/ml Hoechst 33342 (Life technologies) for nuclear staining. Microscopic images were obtained by using a Leica TCS SP5 DMI6000 CS Confocal Microscope (Leica), equipped with ultraviolet 405 Diode, Argon, DPS3561, HeNe594 Lasers. Fluorescent images were captured with a 63.0 × lens zoomed in 1-3× with a 1,024×1,024 frame and 400 Hz scanning speed. For multicolor experiments, the following wavelength settings were used; mCherry (Ex 594 nm/Em 604-766 nm), EGFP (Ex 488 nm/Em 498-567 nm), YFP (Ex 514 nm/Em 520-590 nm), dsRed (Ex 561 nm/Em 571 -723 nm), and Hoechst (Ex 405 m/Em 414-475 nm). Images were further analyzed using Leica LAS AF software (Leica) and Image J (NIH).
3
Imaging Tubulin and DNA in siADAR1-transfected HeLa Cells
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Transfection of siRNAs (siADAR1-1) into HeLa cells at 1 nM concentration was carried out as described above. After incubation for 24 h, the culture medium was replaced with a fresh medium containing CellLight Tubulin-GFP and BacMam 2.0 (Thermo Fisher Scientific). Nuclei were stained with SiR-DNA reagent (Cytoskeleton) at 0.25 μM for 6 h. Cells were cultured on Ibidi μDish 3.5 cm. After 72 h, cells were fixed with 4% paraformaldehyde and soaked in Dulbecco’s PBS. Microscopic images were obtained by using a Leica TCS SP5 DMI6000 CS Confocal Microscope and LAS X software (Leica), equipped with ultraviolet 405 diode, Argon, DPS3561, and HeNe594 lasers. Fluorescent images were captured with a 40× lens with a 512 × 512 frame. For multicolor experiments, the following wavelength settings were used: Tubulin-GFP (Ex 488 nm/Em 498–630 nm) and SiR-DNA reagent (Ex 647 nm/Em 657–800 nm). Nuclear morphological analysis was performed using 4′,6-diamidino-2-phenylindole (DAPI)-stained HeLa cells.
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