For EdU incorporation assays, cells were typically seeded in multiwell black, optically clear bottom, tissue culture–treated plates (PerkinElmer) for imaging and treated with 10 μM EdU (Thermo Fisher Scientific) for the indicated times. Cells were fixed in 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) for 15 min and then permeabilized with 0.5% Triton X-100. Fixed cells were washed with PBS and stained with 1 μM Alexa Fluor 488 or Alexa Fluor 555–conjugated azide diluted in PBS containing 2 mM CuSO4 and 50 mM ascorbic acid. To counterstain the DNA, Hoechst 33342 was added to 2 μg/ml. Cells were incubated for several hours or overnight at room temperature protected from light and evaporation and then washed three times with PBS. Later they were subjected to imaging using Nikon TiE-Eclipse epifluorescence microscope (60 × oil immersion objective) and/or Attune flow analyzer.
Tie eclipse epifluorescence microscope
Manufactured by Nikon
Sourced in Canada
The TiE-Eclipse epifluorescence microscope is a high-performance laboratory instrument designed for advanced imaging applications. It features a modular optical system that allows for the observation and analysis of fluorescently labeled specimens. The microscope provides researchers with the tools necessary to capture detailed, high-quality images for a variety of scientific investigations.
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Lab products found in correlation
Flow analyzer, by Nikon (2 mentions)
5 ethynyl 2 deoxyuridine (edu), by PerkinElmer (2 mentions)
5 ethynyl 2 deoxyuridine (edu), by Thermo Fisher Scientific (2 mentions)
Vectashield containing 4 6 diamidino 2 phenylindole dapi, by Vector Laboratories (2 mentions)
Karyomax colcemid solution, by Thermo Fisher Scientific (2 mentions)
Prolong gold mounting medium, by Thermo Fisher Scientific (1 mentions)
6 protocols using tie eclipse epifluorescence microscope
1
EdU Incorporation Assay Protocol
2
Cell Cycle Synchronization and Microscopy
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Approximately 200 µl of cell cycle synchronized samples was collected and fixed for 15 min at room temperature in 100 µl of 4% paraformaldehyde made in 1× PBS. After fixation, samples were washed once with 500 KPO4/sorbitol buffer (final concentrations of 0.1 M potassium phosphate and 1.2 M sorbitol) and stored in 500 µl of KPO4/Sorbitol buffer at 4°C. Prior to imaging, samples were washed once with 1× PBS and stained with a final DAPI concentration of 1.25 µg/ml in 1× PBS. Arrested cells were imaged on a Nikon TiE-Eclipse epifluorescence microscope (60× oil immersion objective) and a single image capturing the middle of cells was used to assess the presence of a bud and nuclear division status. Cells with DAPI-stained nuclei clearly segregated to mother and daughter cells were categorized as anaphase cells. Between 16 and 145 cells were counted for each time point.
3
EdU Incorporation Assay Protocol
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For EdU incorporation assays, cells were typically seeded in multiwell black, optically clear bottom, tissue culture–treated plates (PerkinElmer) for imaging and treated with 10 μM EdU (Thermo Fisher Scientific) for the indicated times. Cells were fixed in 4% paraformaldehyde (PFA)/phosphate-buffered saline (PBS) for 15 min and then permeabilized with 0.5% Triton X-100. Fixed cells were washed with PBS and stained with 1 μM Alexa Fluor 488 or Alexa Fluor 555–conjugated azide diluted in PBS containing 2 mM CuSO4 and 50 mM ascorbic acid. To counterstain the DNA, Hoechst 33342 was added to 2 μg/ml. Cells were incubated for several hours or overnight at room temperature protected from light and evaporation and then washed three times with PBS. Later they were subjected to imaging using Nikon TiE-Eclipse epifluorescence microscope (60 × oil immersion objective) and/or Attune flow analyzer.
4
Chromosome Spread Preparation and Analysis
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Chromosome spreads were obtained by arresting cells in mitosis in Karyomax Colcemid solution (1:100; Life Technologies) for 4–8 h, followed by harvesting using trypsin and/or accutase. Trypsinized cells were collected by centrifugation for 5 min at 300 × g and gently resuspended in a small amount of medium (~1 ml). Resuspended cells were allowed to swell for 7–10 min in 0.4% KCl solution at room temperature and prefixed by addition of freshly prepared methanol: acetic acid (3:1) fixative solution (~100 μL per 10 mL of total volume). Prefixed cells were collected by centrifugation and fixed in methanol:acetic acid (3:1) fixative solution. Spreads were dropped on a glass slide and incubated at 65°C for at least 1 h. For counting, spreads were mounted in Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA), imaged on a Nikon TiE-Eclipse epifluorescence microscope (60 × oil immersion objective), and counted using the Cell Counter plug-in in ImageJ (Schneider et al., 2012 (link))
5
Chromosome Spread Preparation and Analysis
6
Immunofluorescence Staining of Cytoskeleton
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Cells were seeded on glass coverslips and allowed to adhere overnight. The media was removed and the cells washed with PBS and fixed in 3.7% paraformaldehyde at room temperature for 5 min. Permeabilization was achieved using PBS containing 1% BSA + 0.5% Triton X-100 for 1–2 min at room temperature. Blocking was performed using 1% BSA + 0.05% Triton X-100 in PBS for 30 min followed by incubation with primary antibody for 1 h. Primary antibodies were used at the following dilutions: α-tubulin – 1:1,000, γ-tubulin – 1:500, β-catenin – 1:200, phospho-β-catenin – 1:200, E-cadherin – 1:200. After washing with PBS, cells were incubated in secondary antibody for 1 h. The cells were washed with PBS and stained with Hoechst 33258 at a dilution of 1:5,000 from a 5 mg/ml stock solution. The cells were again washed with PBS and mounted in Prolong Gold mounting medium (Invitrogen). The slides were allowed to dry overnight before imaging using a Nikon TiE Eclipse epifluorescence microscope.
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