CometAssay Electrophoresis System II

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 2 × 10^4 cells/ml/gel	COMET assay included ketoprofen (KP, 10 μM) as positive control. Cells to be pretreated with KP at 37 °C in a CO2 incubator (100% relative humidity and dark conditions) for 30 minutes before the irradiation step.	The percentage of DNA damage of each sample could be calculated with the visual scoring of at least 100 DNA Comets using the following equation: [(Nclass 0 Comets × 0) + (Nclass 1 Comets × 1) + (Nclass 2 Comets × 2) + (Nclass 3 Comets × 3) + [(Nclass 4 Comets × 4) + (Nclass 5 Comets × 5) + (NClass 6 Comets × 6)]/6, where class 0 comets indicate comets with no DNA damage and class 6 comets indicate comets with maximum DNA damage.

Upstream tips
Seed 2 × 10^4 cells/ml/gel
Protocol tips
COMET assay included ketoprofen (KP, 10 μM) as positive control. Cells to be pretreated with KP at 37 °C in a CO2 incubator (100% relative humidity and dark conditions) for 30 minutes before the irradiation step.
Downstream tips
The percentage of DNA damage of each sample could be calculated with the visual scoring of at least 100 DNA Comets using the following equation: [(Nclass 0 Comets × 0) + (Nclass 1 Comets × 1) + (Nclass 2 Comets × 2) + (Nclass 3 Comets × 3) + [(Nclass 4 Comets × 4) + (Nclass 5 Comets × 5) + (NClass 6 Comets × 6)]/6, where class 0 comets indicate comets with no DNA damage and class 6 comets indicate comets with maximum DNA damage.

Publication protocol

Irradiated and non-irradiated cell suspensions (100 μL) were carefully mixed with 100 μL of 1% low melting point agarose, and the drops were loaded on Trevigen treated slides placed on ice until jellification (2 × 104 cells/gel). Then, slides were immediately sub-jected to cell lysis by incubation overnight in cold lysis buffer (2.5 M NaCl, 0.1 M Na2EDTA, 0.01 M Tris, 1% Triton X-100) or maintained in DMEM medium at 37 °C for 3 h and 6 h, and then lysed, to promote intrinsic cellular DNA-repair mechanisms. The next day, all slides were placed in the electrophoretic tank, covered with cold alkaline buffer (0.2 M NaOH, 1 mM EDTA, pH ≥13) and left during 40 min for DNA unwinding. Afterwards, the electrophoresis was carried out at 21 V (1 V/cm) for 30 minutes at 4 °C. Once the electrophoresis ended, the slides were washed twice in PBS for 5 min. DNA was fixed by two subsequent incubations in 70% ethanol and 100% ethanol. DNA comets and tails were stained with SYBR Gold (1:10.000) for 30 min (λexc: 495 nm and λem: 537 nm). Finally, the slides were air-dried and kept in darkness until their visualization. Visualization of comet nucleoids and tails was carried out with a Leica DMI 4000B fluorescence microscope using the Fluorescein FITC filter. At least 5 pictures of each sample were taken in order to determine DNA damage.
The percentage of DNA damage of each sample was calculated with the visual scoring of at least 100 DNA Comets using the following equation: [(Nclass 0 Comets × 0) + (Nclass 1 Comets × 1) + (Nclass 2 Comets × 2) + (Nclass 3 Comets × 3) + [(Nclass 4 Comets × 4) + (Nclass 5 Comets × 5) + (NClass 6 Comets × 6)]/6, where class 0 comets indicate comets with no DNA damage and class 6 comets indicate comets with maximum DNA damage

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