In this study, the prostate cancer cell-line LNCaP was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The cell was cultured in RPMI 1640 medium supplemented with 10% FBS, and 1% penicillin-streptomycin at 37°C in a 5% CO2 incubator. Cells in a logarithmic growth phase were used in all experiments. TUNEL staining was used to determine the antitumor effect by detecting the apoptotic cells in tumor tissues after different treatments. Firstly, seven 35mm glass substrate/confocal culture dishes were inoculated with 105/180µL cells, and 20µL test solution (saline, 50uCi 32P, 2.5μg DOX, 50uCi 32P-FA-PEG-nHA, 100uCi 32P-FA-PEG-nHA, 150uCi 32P/FA-PEG-nHA or 150uCi/2.5ug 32P/DOX-FA-PEG-nHA) was added. Secondly, cells were fixed using 4% paraformaldehyde for 10 min at room temperature after 72h incubation. Then, 200µL PBS solution containing 3% Triton X-100 was added for 10 min at room temperature to break the cell membrane. Finally, a 50 μL TUNEL (C1090, Beyotime, Shanghai, China) reaction mixture (45 fluorescent labeling solution and 5 μL TdT enzyme) was incubated at 37 °C for 60 min in a dark humidified environment. Finally, the nuclei were stained with DAPI (C1005, Beyotime, Shanghai, China) for 5 min at 37 °C, and apoptotic cells were observed by the fluorescence microscope (OPTIKA, Italy).
Fluorescence microscope
Manufactured by Optika
Sourced in Italy
The Fluorescence Microscope is a specialized optical instrument used to observe and analyze samples that exhibit fluorescent properties. The core function of this device is to illuminate the sample with a specific wavelength of light, causing the fluorescent molecules within the sample to emit light at a different wavelength, which is then detected and magnified for visual observation.
Automatically generated - may contain errors
Lab products found in correlation
C1005, by Beyotime (1 mentions)
C1090, by Beyotime (1 mentions)
Lncap, by National Collection of Authenticated Cell Cultures (1 mentions)
Ab7260, by Abcam (1 mentions)
Triton x 100, by Solarbio (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Paraformaldehyde (pfa), by Solarbio (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
7 protocols using fluorescence microscope
1
Evaluating Apoptosis in Prostate Cancer
2
Mitochondrial Membrane Potential Assay
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Changes in the mitochondrial transmembrane potential were studied as described by Chen et al. (55 (link)). Cells of the test isolate were grown in Sabouraud dextrose broth to log phase and then treated with different concentrations of GQAs (2, 4, or 8 μg/mL) for 6 h at 37°C with shaking. After incubation, cells were harvested, washed with PBS (pH 7.4), and then suspended in HEPES buffer (10 mM, pH 7.4) containing 100 nM rhodamine B (Rho-B) and 5% glucose. The cells were washed, and fluorescence was measured using flow cytometry with an excitation wavelength of 555 nm and emission wavelength of 579 nm. Additionally, Rho-B-stained cells of Candida were visualized with a fluorescence microscope (Optika, Italy).
3
Mitochondrial Membrane Potential Assay
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Changes in the mitochondrial transmembrane potential were studied as described by Chen et al. (55 (link)). Cells of the test isolate were grown in Sabouraud dextrose broth to log phase and then treated with different concentrations of GQAs (2, 4, or 8 μg/mL) for 6 h at 37°C with shaking. After incubation, cells were harvested, washed with PBS (pH 7.4), and then suspended in HEPES buffer (10 mM, pH 7.4) containing 100 nM rhodamine B (Rho-B) and 5% glucose. The cells were washed, and fluorescence was measured using flow cytometry with an excitation wavelength of 555 nm and emission wavelength of 579 nm. Additionally, Rho-B-stained cells of Candida were visualized with a fluorescence microscope (Optika, Italy).
4
Astrocyte Identification via GFAP Immunofluorescence
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The glial fibrillary acidic protein (GFAP) expression in astrocytes was identified using immunofluorescence staining. In short, we cultured the cells at a density of 4 × 10 5 cells/ml in poly-l-lysinecoated coverslips. After attaching the cells to the flask, immunofluorescence staining was applied to identify astrocytes. Fresh 4% paraformaldehyde (Solarbio, Beijing, China) was prepared for fixing the cells at 4°C for 30 min. After that, we washed the cells once using phosphate-buffered saline (PBS) containing 5% penicillin/streptomycin (Corning Inc., Corning, NY, USA) and the cells were lysed using 0.1% Triton X-100 (Solarbio, Beijing, China) at room temperature for 20 min. Then, we washed the cells with PBS three times, and blocked using 3% BSA in PBS for 1 h, and combined with the primary antibody against GFAP (rabbit anti-GFAP, 1:800; ab7260, Abcam, Cambridge, UK). The cells were incubated overnight in a humid room at 4°C. The cells were washed with PBS three times, and then were incubated with donkey anti-rabbit IgG-488 (Sigma-Aldrich, MO, USA) at 37°C for 1.5 h in the dark and stained with 4 0,6-diamidino-2-phenylindole (Cell Signaling Technology, Danfoss, Massachusetts, USA). All images were obtained through a fluorescence microscope (Optika, Ponteranica, Bergamo, Italy) with appropriate filters.
5
Cytotoxicity Evaluation of Mlt, ZnO/Mlt, and ZnO/Mlt/Chsn
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To evaluate cytotoxicity, L929 cells (Pasteur Institute, Tehran, Iran) (1 × 105–1 × 106 cells/mL) were investigated as previously reported by Tominaga et al., (1999) using staining with calcein-AM and a fluorescence microscope (Optika, Italy) with 490 nm excitation to monitor viable cells for Mlt, ZnO/Mlt and ZnO/Mlt/Chsn38 (link).
6
Apoptosis Evaluation: Morphological Changes
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Cell shrinkage, membrane blebbing, and formation of apoptotic bodies were evaluated for 12 h (high magnification 400×) using a light microscope (Nikon Eclipse TS100; Tokyo, Japan). Cells undergoing apoptosis exhibit an increase in chromatin condensation. Morphologically, the apoptotic nuclei become smaller compared to those of normal cells; also, they become hyperfluorescent when labeled with some nuclear stains. Cells (2×104 cells/mL in a 24-well tissue culture plate) were either left untreated or stimulated with test compound (10, 20, 30, and 40 µg/mL) for 12 hours. The nuclear morphology was observed under fluorescence microscope (Optika Microscopes, Ponteranica, Italy) using respective filters.
7
Autophagy Induction in HepaRG Cells
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HepaRG were seeded on a glass coverslip in a 6 well-plate. Using the Neon electroporator, cells were co-transfected with the LC3-GFP plasmid together with plasmids encoding scramble or Gβ5 shRNA. 16 h after transfection, cells were challenged with APAP (5 mM) for 6 h. Post treatment, cells were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 2 h at room temperature, mounted with Vectashield and DAPI (Invitrogen) and kept at −20 °C for microscopy. Pictures were taken using a fluorescence microscope (Optika, Italy) with a 40× objective. >15 cells/coverslip were counted randomly to quantify the autophagic puncta per cell. The LC3-GFP plasmid was the kind gift from Dr. Santosh Chauhan, Institute of Life Science, Bhubaneswar, India.
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