HCC cells seeded on microscope cover glass were transfected with the mRFP-GFP-LC3 adenovirus (Hanbio; Shanghai, China) for 24 h. Then cells were transfected with miR-223 mimic, miR-223 inhibitor, or FOXO3a siRNA, and treated with doxorubicin (0.25 μg/ml) for 48 h. After the above procedure, cells were fixed with 4% paraformaldehyde, and images were obtained using a TCS SP8 MP laser scanning confocal microscope (Leica; Wetzlar, Germany). Autophagy flux was then assessed by confocal counting of the GFP positive/mRFP positive (yellow) and GFP negative/mRFP positive (red) puncta in cells. 50–100 cells were counted per group in triplicate experiments.
Tcs sp8 mp laser scanning confocal microscope
Manufactured by Leica
Sourced in Germany
The Leica TCS SP8 MP is a laser scanning confocal microscope designed for advanced imaging applications. It features multi-photon excitation capabilities, enabling deep tissue imaging. The system provides high-resolution, 3D imaging and is suitable for a range of scientific research applications.
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3 protocols using tcs sp8 mp laser scanning confocal microscope
1
Autophagy flux quantification in HCC cells
2
Visualization of Mitochondrial Calcein
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8505C cells were seeded into a 35‐ mm glass bottom culture dish. The cells were resuspended in PBS and loaded with calcein AM staining solution (Beyotime Biotechnology, NO. C2009S) and 3 × CoCl2 in the dark at 37°C for 30–45 min. At the end of incubation, culture medium was replaced with medium preheated to 37°C, and the dishes were incubated at 37°C for 30 min in order to ensure that calcein AM was hydrolyzed by lactamase to produce green fluorescent calcein. The cells were washed with PBS 2–3 times, and the fluorescence image of mitochondrial calcein were acquired using a Leica TCS SP8 MP laser scanning confocal microscope.
3
Mitochondrial Calcein Fluorescence Assay
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The cells were trypsinized and resuspended in PBS, then loaded with calcein AM and cytosolic calcein fluorescence quencher (CoCl2) for 15 min at 37 °C. The cells were centrifuged, washed, and the cell pellets were resuspended in PBS and characterized for mitochondrial calcein fluorescence by flow cytometry analysis.
The cells were loaded with calcein-AM and a cytosolic calcein fluorescence quencher (CoCl2) for 30 min at 37 °C after the H2O2 treatment. Mitochondrial calcein fluorescence images were acquired with a Leica TCS SP8 MP laser scanning confocal microscope.
The fluorescence intensity was calculated as a percentage by gating upon the positive and negative controls.
The cells were loaded with calcein-AM and a cytosolic calcein fluorescence quencher (CoCl2) for 30 min at 37 °C after the H2O2 treatment. Mitochondrial calcein fluorescence images were acquired with a Leica TCS SP8 MP laser scanning confocal microscope.
The fluorescence intensity was calculated as a percentage by gating upon the positive and negative controls.
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