Alkali comet assay was performed using Comet Assay Single Cell Gel Electrophoresis Assay Kit (Cat. 4250-050-K, Trevigen) according to the manufacturer’s protocol. Cells were mixed with low-melting agarose before plating on comet assay slides for overnight lysis. The next day, chromosomal DNA was denatured under alkali unwinding buffer (pH>13) and underwent electrophoresis (20V) for one hour at 4°C. After drying the slides, DNA was stained with SYBR Gold (Cat. S11494, ThermoFisher) and images were taken with a FITC filter using a Zeiss fluorescence microscope (Zeiss, San Diego, CA, USA) then analyzed by Open Comet Assay using Image J application as described previously (45 (link)).
Comet assay single cell gel electrophoresis assay kit
Manufactured by Bio-Techne
The Comet Assay Single Cell Gel Electrophoresis Assay Kit is a laboratory tool used to measure DNA damage and repair in individual cells. The kit provides the necessary reagents and components to perform the comet assay, a widely used technique for evaluating DNA strand breaks and alkali-labile sites.
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2 protocols using comet assay single cell gel electrophoresis assay kit
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Alkali Comet Assay for DNA Damage
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Comet Assay for DNA Damage Assessment
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The Comet Assay Single-Cell Gel Electrophoresis Assay Kit (Trevigen) was used according to the manufacturer’s protocol. Briefly, cells were irradiated with or without 10 Gy of IR and recovered for indicated time at 37°C. Cells were collected and rinsed twice with ice-cold PBS; 1 × 105/ml cells were combined with 1% low melting (LM) Agarose at 37°C at the ratio of 1:10 (v/v) and immediately pipetted onto slides. For cellular lysis, the slides were immersed in the lysis buffer overnight at 4°C. Then, the slides were subjected to electrophoresis at 21 V for 45 min and stained in SYBR for 20 min. All images were taken with a fluorescence microscope and analyzed by ImageJ with OpenComet.
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