Chromosomes spreads were prepared as described previously (Lemaçon et al., 2017 (link)). Briefly, 48 h after siControl or siTDP-43 treatment, SH-SY5Y cells were treated with 10 μM nocodazole for 4 h. Arrested cells were trypsinized and resuspended in warm hypotonic solution (75 mM KCl, 5% FBS) for 10 min at 37°C. Then, 500 μl of 4°C fixing solution (3:1 ethanol:acetic acid) was added to cells while gently vortexing. Fixed cells were washed three times with 4°C fixation buffer and stored at 4°C for at least 24 h before spreading. Chromosomes were spread by dropping onto chilled glass slides. Slides were air dried and mounted with Prolong Gold Antifade (Invitrogen) and DAPI. Images were acquired with a fluorescence microscope (Leica DMU 4000B; 63×/1.40-0.60 NA oil). Captured images were analyzed with ImageJ. At least 50 metaphase spreads were assessed per sample. Chromosomes were assessed for both chromatid breakage and end-to-end fusions. For each condition, the total number of aberrations counted was divided by the total number of metaphase spreads assessed to determine the average number of aberrations per cell.
Dmu 4000b
Manufactured by Leica
The DMU 4000B is a high-precision coordinate measuring machine developed by Leica. It is designed to perform accurate dimensional measurements on a variety of workpieces. The DMU 4000B utilizes advanced sensor technology and software to provide reliable and repeatable measurement results.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold antifade, by Thermo Fisher Scientific (3 mentions)
Dfc345 fx camera, by Leica (3 mentions)
Cometslide, by Bio-Techne (2 mentions)
Sybr gold nucleic acid gel stain, by Thermo Fisher Scientific (1 mentions)
Cometassay, by Bio-Techne (1 mentions)
Lmagarose, by Bio-Techne (1 mentions)
Dfc345 fx, by Leica (1 mentions)
Lysis solution, by Bio-Techne (1 mentions)
6 protocols using dmu 4000b
1
Chromosomal Aberration Quantification
2
Neutral Comet Assay for DNA Damage
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Neutral comet assay was performed as previously described (40 (link)). Briefly, 700 cells were resuspended in 70 μl 0.5% low melting point agarose (Trevigen, Cat. No. 4250-050-02) and spread on a comet slide (Trevigen, Cat. No. 4250-200-03). Cells were lysed in a cold lysis solution (Trevigen, Cat. No. 4250-050-01) at 4°C for 30 min. DNA migration was performed in TBE buffer at 1 V/cm for 30 min. Slides were washed in milliQ water, fixed with ethanol 70% for 30 min and dried at room temperature. Comets were labeled with SYBR® Gold Nucleic Acid Gel Stain (ThermoFisher) for 30 min. Images were acquired with a fluorescence microscope (LEICA DMU 4000B; 20×/0.4 CORR) coupled to the LEICA DFC345FX camera. The images were analyzed using the OpenComet plug-in from ImageJ. At least 100 comets were scored per sample in each experiment. Olive moment measures DSBs as a product of the amount of DNA present (intensity) times the size of the broken DNA pieces (length) in the tail.
3
Nocodazole-Induced Chromosome Spread Analysis
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Cells were treated with 10 μM nocodazole for 4 h. Cells were collected, washed and resuspended in 10 ml of warmed hypotonic solution (10 mM KCl, 10% FBS) for 10 min at 37°C. Cells were fixed by adding 500 μl of cold fixation buffer (acetic acid:ethanol 1:3). Cell pellets were washed four times with the cold fixation buffer and stored in this buffer at 4°C overnight. The nuclei were spread on cold slides. The slides were air dried overnight and mounted with Prolong Gold Antifade (Invitrogen) with DAPI. Images were acquired with a fluorescence microscope (LEICA DMU 4000B; 63×/1.40–0.60 NA oil) coupled to the LEICA DFC345FX camera. The images were analyzed with ImageJ. At least 50 metaphases per sample were scored in each experiment. Statistical significance was assessed using unpaired non-parametric Man–Whitney compared ranks t-test.
4
Comet Assay Protocol for DNA Damage Analysis
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Neutral comet assays were performed using CometAssay (Trevigen) according to the manufacturer's protocol with minor modifications (Carvajal-Maldonado et al., 2019 (link)). Upon harvesting, cells were suspended at 3×105 cells/ml in cold PBS before combining with LMAgarose (Trevigen, 4250-50-050-02), spread onto a comet slide (Trevigen, 4250-200-03) and allowed to dry. Slides were placed in lysis solution (Trevigen, 4250-050-01) at 4°C for 1 h. Lysed slides were immersed in 1× TBE buffer (0.1 M Tris base, 0.1 M boric acid, 2.5 mM EDTA) for 30 min before electrophoresis at 25 V for 30 min at 4°C. Slides were washed in DNA precipitate solution (1 M ammonium acetate, 95% EtOH) for 30 min, followed by a fixing step in 70% ethanol for 30 min, and dried overnight at RT. Comets were stained with 1× SYBR Gold (Thermo Fisher Scientific) for 30 min. Images were acquired with a fluorescence microscope (Leica DMU 4000B; 63×/1.40-0.60 NA oil) with a Leica DFC345FX camera. At least 150 comets were scored in each experiment using the OpenComet plugin in the ImageJ analysis software.
5
Quantitative DNA Fiber Imaging Analysis
6
HU-Nocodazole Synchronization and Metaphase Analysis
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Cells were treated with 4 mM HU for 6 h, washed twice with PBS, and released for 24 h in fresh medium. During the last 4 h, 10 μM nocodazole was added to the medium. Cells were collected, washed and resuspended in 10 ml of warmed hypotonic solution (10 mM KCl, 10% FBS) for 10 min at 37 °C. Cells were fixed by adding 500 μl of cold fixation buffer (acetic acid 1: 3 ethanol). Cell pellets were washed four times with the cold fixation buffer and stored in this buffer at 4 °C overnight. The nuclei were spread on cold slides. The slides were air dried overnight and mounted with Prolong Gold Antifade (Invitrogen) with DAPI. Images were acquired with a fluorescence microscope (LEICA DMU 4000B; 63×/1.40-0.60 NA oil) coupled to the LEICA DFC345FX camera. The images were analyzed with ImageJ. At least 50 metaphases per sample were scored in each experiment.
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