HeLa or SiHa cells were seeded in 6-well plates at a density of 1,000 cells/well. Cells were then cultured at 37°C for 10 days until visible colonies appeared. Colonies were stained with 500 µl Giemsa solution (Nanjing KeyGen Biotech Co., Ltd.) and incubated for 30 min at 37°C. Colonies were then imaged using a X71 (U-RFL-T) fluorescence microscope (Olympus Corporation; magnification, ×40).
X71 u rfl t fluorescence microscope
Manufactured by Olympus
Sourced in United States, Japan
The X71 (U-RFL-T) is a fluorescence microscope designed for laboratory use. It features a fluorescence illumination system and is capable of observing specimens that have been labeled with fluorescent markers.
Automatically generated - may contain errors
Lab products found in correlation
Crystal violet, by Merck Group (4 mentions)
Matrigel, by BD (4 mentions)
Matrigel, by Merck Group (2 mentions)
Vectashield, by Vector Laboratories (2 mentions)
Synergy 2 multi mode microplate reader, by Agilent Technologies (2 mentions)
Triton x 100, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Normal goat serum (ngs), by Merck Group (2 mentions)
Giemsa solution, by Keygen Biotech (1 mentions)
Transwell insert, by Corning (1 mentions)
Jc 1 staining, by Thermo Fisher Scientific (1 mentions)
H2dcfda, by Merck Group (1 mentions)
Jc 1 probe, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Edu cell proliferation assay kit, by RiboBio (1 mentions)
Mitosox reagent, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Vector Laboratories (1 mentions)
Ab93876, by Abcam (1 mentions)
Ab23981, by Abcam (1 mentions)
61 protocols using x71 u rfl t fluorescence microscope
1
Clonogenic Assay for Cell Viability
2
Invasion Capacity Assay with Matrigel
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In order to analyze invasion capacity, 1x104 cells were suspended and plated into the upper chamber of Transwell inserts (8-µm pore size; Corning Inc.), coated with 0.3% Matrigel (Sigma-Aldrich; Merck KGaA) in RPMI-1640 medium without FBS addition, and maintained in an incubator at 37˚C. In the lower chamber, 500 µl RPMI-1640 medium supplemented with 10% FBS was added. After 24-h incubation, the invaded cells were fixed with 4% paraformaldehyde at room temperature for 10 min and stained with 0.25% crystal violet (Sigma-Aldrich; Merck KGaA) at room temperature for 10 min. Invaded cells were then imaged under a X71 (U-RFL-T) fluorescence microscope (Olympus Corporation; magnification, x40).
3
Evaluating Cell Migration and Invasion
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Cell migration and invasion was evaluated using an 8-mm pore size Transwell system (Costar; Corning, Inc.) without or with Matrigel (BD Biosciences), which was pre-coated at 37˚C for 2 h. Briefly, cells were dissociated into single cells and resuspended in DMEM medium at a density of 1x105 cells/ml. The top chamber of the Transwell was loaded with 200 µl cell suspension and 800 µl DMEM medium supplemented with 10% FBS was added to each lower chamber. Following incubation in the incubator for 24 h at 37˚C, the cells remain on the upper surface of upper chamber were removed and the cells on the lower surface of upper chamber were fixed in 4% paraformaldehyde at room temperature for 10 min and subsequently stained with 0.25% crystal violet (MilliporeSigma) at room temperature for 10 min followed by three washes with PBS. Images of the stained cells from five random views were captured under a X71 (U-RFL-T) fluorescence microscope (Olympus Corporation) at x20 magnification.
4
Cisplatin Cytotoxicity Assay via Clonogenic Assay
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The clonogenic assay, which is used to determine the effectiveness of cytotoxic agents, including chemotherapeutic agents, was performed to determine the sensitivity of cells to cisplatin. Cells (1×105) were seeded in a 6-well plate and allowed to attach overnight at 37°C, and maintained with 0.25, 0.5, 0.75, 1, 1.5 or 2 µM cisplatin for 14–21 days. Subsequently, the colonies were observed by fixation with 4% paraformaldehyde and staining with methanol (25% v/v) substituted with crystal violet (0.05% w/v) for 30 min, prior to being washed with 1X PBS. Colonies >40 µm in diameter were counted using a X71 (U-RFL-T) fluorescence microscope (Olympus, Melville, NY, USA).
5
Evaluating Mitochondrial Membrane Potential
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JC-1 staining (Thermo Fisher Scientific, Inc.) was used to investigate the mitochondrial membrane potential. Cells were stained with 15 µg/ml JC-1 at 37˚C for 30 min in dark. Subsequently, cells were washed 3 times with PBS. Stained cells were observed using an X71 (U-RFL-T) fluorescence microscope (Olympus Corporation) at a magnification, x100.
6
Intracellular ROS Detection Using H2DCFDA
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The specific fluorescent probe H2DCFDA (Sigma-Aldrich) was used to detect the intracellular ROS level. H2DCFDA (5 µl) was added for 15 min at 25°C. The cells in 6-well plates were washed three times with PBS and imaged using an X71 (U-RFL-T) fluorescence microscope (Olympus Corporation, magnification, ×40).
7
Measuring Mitochondrial Membrane Potential
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JC-1 probe (Sigma-Aldrich; Merck KGaA) was used to investigate the mitochondrial membrane potential following erastin treatment. HGC-27 cells were incubated with JC-1 (20 µg/ml) at 37°C for 20 min and then washed twice with PBS. Treated cells were observed by an X71 (U-RFL-T) fluorescence microscope (Olympus Corporation, magnification, ×40).
8
Intracellular and Mitochondrial ROS Quantification
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To measure intracellular and mitochondrial ROS levels, 2',7'-dichlorofluorescin diacetate (DCFH-DA) and MitoSOX™ staining were performed, respectively. Nuclei were counterstained with DAPI to a final concentration of 5 µg/ml (Beyotime Institute of Biotechnology) at room temperature for 5 min. Cells were washed 3 times with ice-cold PBS. Subsequently, cells were incubated with 1 ml serum-free medium containing 10 µM DCFH-DA probe or 5 µM MitoSOX™ in the dark at 37˚C for 30 min with gentle shaking every 5 min. Cells were washed 3 times with ice-cold PBS. Green fluorescence was observed using an X71 (U-RFL-T) fluorescence microscope (Olympus Corporation) at an excitation wavelength of 488 nm and magnification, x40.
9
EdU Cell Proliferation Assay
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Cell proliferation was detected by the incorporation of EdU with an EdU Cell Proliferation Assay kit (Guangzhou RiboBio Co., Ltd.). Briefly, cells were incubated with 50 mM EdU for 4 h and then fixed with 4% paraformaldehyde for 30 min at 25°C. Then, EdU staining was performed according to the manufacturer's protocol. Cell nuclei were stained with Hoechst-33342 (Sigma-Aldrich; Merck KGaA) at a concentration of 1 mg/ml for 20 min. The proportion of cells incorporating EdU was determined using an X71 (U-RFL-T) fluorescence microscope (Olympus Corporation; magnification, ×40).
10
Transwell Invasion Assay for Cell Migration
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Cell invasion was evaluated using 8‐mm pore Transwell inserts (Costar; Corning, Inc.) which were precoated with Matrigel (BD Biosciences) at 37°C for 2 h. Briefly, cells were dissociated into single cells and resuspended in DMEM at a density of 1 × 105 cells/ml. A quantity of 200 μl cell suspension was added to the upper chamber, and 800 μl DMEM supplemented with 10% FBS was added to the lower chamber. Following incubation for 24 h at 37°C, cells on the upper surface of the upper chamber were removed, and cells on the lower surface of the upper chamber were fixed in 4% paraformaldehyde at room temperature for 10 min. Cells were subsequently stained with 0.25% crystal violet (MilliporeSigma) at room temperature for 10 min, followed by three washes with PBS. Images of stained cells from five random views were captured under an X71 (U‐RFL‐T) fluorescence microscope (Olympus Corporation; magnification, ×20).
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