To detect DNA double-strand breaks (DSBs), neutral comet assays were performed as previously described [29] (link). Shortly, cells were harvested, 8000 cells were diluted in 400 µl PBS and embedded in 1.2 ml 1% low-gelling agarose (Sigma). 100 µl of the cell suspension was used to make gels onto Trevigen comet assay slides. To lyse the cells in the gel, slides were incubated in lysis solution (2% sarkosyl, 0.5 M Na2EDTA and 0.5 mg/ml Proteinase K) overnight at 37 °C. The next day, slides were rinsed three times for 30 min at room temperature in electrophoresis buffer (90 mM TrisHCl pH = 8.5, 90 mM Boric Acid and 2 mM Na2EDTA). Electrophoresis was performed for 25 min at 20 V in electrophoresis buffer. Subsequently, slides were washed once with distilled water. To stain DNA slides were incubated with 2.5 μg/ml Propidium Iodide diluted in distilled water for 20 min. Individual comets were imaged with a Zeiss AxioObserver Z1 inverted microscope using a 20x objective equipped with a Hamamatsu ORCA AG Black and White CCD camera. Tailmoments of individual comets were assessed using the CASP software (http://casplab.com/ ). For each condition, more than 50 cells were analyzed.
Black and white ccd camera
Manufactured by Hamamatsu Photonics
The Black and White CCD camera is a high-performance imaging device designed for various applications. It utilizes a charge-coupled device (CCD) sensor to capture monochrome images with exceptional clarity and precision. The camera offers reliable and consistent image acquisition capabilities, making it a versatile tool for a wide range of scientific and industrial needs.
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3 protocols using black and white ccd camera
1
Neutral Comet Assay for DNA Breaks
2
DNA Replication Dynamics Quantification
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Cells were pulse-labelled with 25 μM CldU followed by 250 μM IdU for 20–40 min each. After labelling, cells were trypsinized and lysed in spreading buffer (200 mM Tris-Hcl pH 7.4, 50 mM EDTA and 0.5% SDS) before spreading on a microscope slide (Menzel-Gläser,Superfrost). Slides were fixed in methanol: acidic acid 3:1. Before immunodetection, slides were treated with 2.5 M HCl for 1 hr and 15 min. To detect CldU and IdU labelled tracts slide were incubated for 1 hr with rat-anti-Brdu (Clone BU1/75, Novus Biologicals; 1:500) and mouse-anti-BrdU (clone B44, Becton Diskinson; 1:750), respectively. Subsequently, slides were fixed with 4% paraformaldehyde for 10 min and incubated with Alexa 488-labeled goat-anti-mouse and Alexa 555-labeled goat-ant-rat (Molecular probes; 1:500) for 1 hr and 30 min. Pictures were taken with a Zeiss AxioObserver Z1 inverted microscope using a 63x lens equipped with a cooled Hamamatsu ORCA AG Black and White CCD camera and track lengths were analyzed with ImageJ software. Replication track lengths were calculated using the conversion factor 1 μm = 2.59 kb (Jackson and Pombo, 1998 (link)). The 1-way ANOVA (nonparametric Kruskal-Wallis test) was used for statistical analyses.
3
Neutral Comet Assay Protocol
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Neutral comet assays were performed as described by Olive et al. (Olive and Banáth, 2006 (link)). Pictures of individual cells were taken with a Zeiss AxioObserver Z1 inverted microscope equipped with a cooled Hamamatsu ORCA AG Black and White CCD camera and analyzed with CASP software (http://www.casp.of.pl ). The p-value was determined using 1-way ANOVA (nonparametric Kruskal-Wallis test).
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