Cells were pelleted and resuspended in ice-cold PBS. The resuspended cells were mixed with 1.5% low melting point agarose. This mixture was loaded onto a fully frosted slide that had been pre-coated with 0.7% agarose and a coverslip was then applied to the slide. After the gelation of the cell mixture, the coverslip was removed. The slides were then submerged in pre-chilled lysis solution (1% Triton X-100, 2.5 M NaCl, and 10 mM EDTA, pH 10.5) for 30 min at 4°C. After soaking with pre-chilled unwinding and electrophoresis buffer (0.3 N NaOH and 1 mM EDTA) for 30 min, the slides were subjected to electrophoresis for 15 min at 0.5 V/cm (25 mA). After electrophoresis, slides were stained with 1X SYBR Gold (Molecular Probes) and nuclei images were visualized and captured at 200X magnifications with a Zeiss AxioImager A1 fluorescent microscope (Zeiss, Germany) equipped with a CCD camera (Optronics, Goleta, CA). Over one hundred of cells in each sample were scored to calculate the average of comet tail moment (Tail moment = %DNAtail × Lengthtail) using TriTek CometScoreTM software.
Ccd camera
Manufactured by Optronics
Sourced in Germany
The CCD camera is a type of image sensor that captures light and converts it into digital signals. It is a core component in various laboratory equipment, serving as the primary means of image acquisition and data collection. The CCD camera's function is to transform optical images into electrical signals that can be processed and analyzed by connected systems.
Automatically generated - may contain errors
6 protocols using ccd camera
1
Comet Assay for DNA Damage
2
Comet Assay for CFEZO-Induced DNA Damage
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CFEZO-induced DNA damage was determined using a comet assay. Cells were treated with the indicated concentrations of CFEZO for 24 h in complete medium, harvested, resuspended in ice-cold PBS, and processed under a dimmed light, as described previously30,31 (link). Prepared comet slides were viewed, and nuclei images were visualized and captured at 100× magnification with an Axioplan 2 fluorescence microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a CCD camera (Optronics, Goleta, CA, USA).
3
Podoplanin Immunohistochemistry Protocol
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H&E staining was performed using a standard procedure with a Leica autostainer (ST5010 Autostainer XL, Leica Biosystems, Ontario, Canada). Immunoperoxidase staining of podoplanin (D2-40; Cedarlane Laboratories, Ontario, Canada) was performed as it has been described previously.16 (link) D2-40, a marker of lymphatic vessel endothelium,17 (link) is an antibody that is designed to target a podoplanin protein epitope modified after formalin fixation. Sections were viewed and imaged with an upright microscope (BX51, Olympus, Tokyo, Japan) equipped with a CCD camera (Optronics, California, USA).
4
Comet Assay for DNA Damage
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After treatment, the cells were pelleted and resuspended in ice-cold PBS. The resuspended cells were mixed with 1.5% low melting point agarose. This mixture was loaded onto a fully frosted slide that had been pre-coated with 0.7% agarose and a coverslip was then applied to the slide. The slides were submerged in pre-chilled lysis solution (1% Triton X-100, 2.5 M NaCl, and 10 mM EDTA, pH 10.5) for 1 hour at 4°C. After soaking with pre-chilled unwinding and electrophoresis buffer (0.3 N NaOH and 1 mM EDTA) for 20 minutes, the slides were subjected to electrophoresis for 15 minutes at 0.5 V/cm (20 mA). After electrophoresis, slides were stained with 1X Sybr Gold (Molecular Probes) and nuclei images were visualized and captured at 400X magnifications with an Axioplan 2 fluorescence microscope (Zeiss, Germany) equipped with a CCD camera (Optronics, Goleta, CA). Over hundreds of cells were scored to calculate the overall percentage of comet tail-positive cells.
5
Comet Assay for Quantifying DNA Damage
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Cells were seeded in 12-well plates and treated, collected, and resuspended in ice-cold PBS. Next, the resuspended cells were mixed with 1.5% low-melting point agarose and loaded onto a fully frosted slide precoated with 0.7% agarose. A coverslip was placed on the slide, which was then submerged in prechilled lysis solution (1% Triton X-100, 2.5 M NaCl, and 10 mM EDTA, pH 10.5) for 1 h at 4°C. After soaking in prechilled unwinding and electrophoresis buffer (0.3 M NaOH and 1 mM EDTA) for 20 min, the slides were subjected to electrophoresis for 15 min at 0.5 V/cm (20 mA), stained with 1× Sybr Gold (Molecular Probes), and nuclear images were visualized and captured using an Axioplan 2 fluorescence microscope (magnification, ×400; Carl Zeiss, Jena, Germany) equipped with a CCD camera (Optronics). More than hundreds of cells were scored to calculate the overall comet tail-positive cell percentage.
6
Comet Assay for DNA Damage Analysis
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