Cell death was detected using propidium iodide (PI) (Sigma-Aldrich, USA) and acridine-orange (AO) (Sigma-Aldrich, USA) double staining and examined under fluorescence microscope as previously described by [27 (link)]. Briefly, 1 × 106 MCF-7 cells/ml were plated in 6-well plate. The IC50 concentration were used for all five extracts and DMSO served as the vehicle control (control). Cells were treated for 24, 48 and 72 h. The treated cells were trypsinized and centrifuged at 1000×g for 10 min. After rinsing with PBS, the supernatant was discarded and 10 μl fluorescent dyes, AO (10 μg/ml) and PI (10 μg/ml), were added into the cellular pellet at equal volumes. Stained cells were transferred onto a glass slide and observed under ultraviolet (UV)-fluorescence microscope (Olympus, Japan) within 30 min.
Uv fluorescence microscope
Manufactured by Olympus
Sourced in Japan
The UV fluorescence microscope is an optical microscope that uses ultraviolet light to excite fluorescent molecules within a sample, allowing for the visualization of specific structures or components. The core function of this instrument is to enable the observation and analysis of fluorescently labeled specimens.
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3 protocols using uv fluorescence microscope
1
Fluorescent Staining for Cell Death Analysis
2
Cell Cycle and Apoptosis Analysis
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Cell cycle progression was assayed by measuring the incorporation of 5-bromo-2-deoxyuridine (BrdU) into DNA of cells during the S phase of the cell cycle [82 (link)]. BrdU detection was performed as described previously [80 (link)]. A population of HUVEC cells was analyzed in multiple randomly chosen microscopic fields to determine the BrdU labeling index (percentage of cell synthesizing DNA). Apoptotic cell death was measured by fluorescent microscopic analysis of DNA staining patterns with Hoechst 33258 as previously described [82 (link)]. Apoptotic nuclear morphology was quantified with a UV fluorescence microscope (Olympus). Apoptotic cells were identified by nuclear condensation and fragmentation and including the higher intensity of blue fluorescence of the nuclei.
3
Bergamottin Induces Apoptosis in A549 Cells
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Morphological study by phase contrast microscopy. A549 cells were seeded into 6-well plates at a density of 1x10 6 cells/well in 1 ml medium. The cells were treated with the different concentrations (0, 10, 25 and 50 µM) of bergamottin for 48 h. The morphological changes were observed and the images were captured under an inverted light microscope (Olympus; Olympus Optical Co., Ltd., Tokyo, Japan) after 48 h. The same spot of cells was marked and captured. The images were captured at a magnification of x200.
Morphological study of apoptosis using fluorescence microscopy. A549 cells were seeded into 12-well plates at a density of 1x10 6 cells/well in 1 ml culture medium. After treatment with the different concentrations (0, 10, 25 and 50 µM) of bergamottin for 48 h, cell apoptosis was determined by the Hoechst staining kit according to the manufacturer's instructions. After the treatment, cells were fixed with 4% polyoxymethylene and then incubated in Hoechst solution for 10-15 min in the dark. The staining images were recorded using a UV fluorescence microscope (Olympus) using a UV filter at a magnification of x200 to detect morphological evidence of apoptosis.
Morphological study of apoptosis using fluorescence microscopy. A549 cells were seeded into 12-well plates at a density of 1x10 6 cells/well in 1 ml culture medium. After treatment with the different concentrations (0, 10, 25 and 50 µM) of bergamottin for 48 h, cell apoptosis was determined by the Hoechst staining kit according to the manufacturer's instructions. After the treatment, cells were fixed with 4% polyoxymethylene and then incubated in Hoechst solution for 10-15 min in the dark. The staining images were recorded using a UV fluorescence microscope (Olympus) using a UV filter at a magnification of x200 to detect morphological evidence of apoptosis.
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