DNA damage was assessed by using the CometAssay® reagent kit for single cell gel electrophoresis assay (Trevigen, MD USA), following the recommended protocol for neutral conditions, and adapting the gel electrophoresis methods for use in the Sub-Cell GT electrophoresis system (Bio-Rad, CA USA). Briefly, cells were collected from coverslips by treatment with 0.25% trypsin, pelleted and resuspended at 100,000 cells/ml in 1X DPBS (Ca2+ and Mg2+ free; Thermo Fisher Scientific) and verified to be greater than 95% viable by tryptan blue exclusion using an automated cell counter before continuing analysis. Aproximately 5,000 cells were embedded in low melting agarose, plated on slides and lysed overnight. The next day, electrophoresis was run at 30 Volts for 30 minutes in 1X TBE (National Diagnostics). Samples were fixed in 70% ethanol for 5 minutes, and slides were immersed in 1X TE buffer pH 8.0 (Ambion) with 1:10 of 10,000x SYBR green nucleic acid stain (Thermo Fisher Scientific). Fluorescent images were captured using a Leica DMI 400B inverted microscope for scoring.
Dpbs ca2 and mg2 free
Manufactured by Thermo Fisher Scientific
DPBS (Ca2+ and Mg2+ free) is a phosphate-buffered saline solution that does not contain calcium and magnesium ions. It is commonly used as a medium for cell culture and other in vitro applications where the presence of these ions is not required.
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2 protocols using dpbs ca2 and mg2 free
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DNA Damage Quantification by Comet Assay
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DNA Damage Quantification by Comet Assay
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DNA damage was assessed by using the CometAssay® reagent kit for single cell gel electrophoresis assay (Trevigen, MD USA), following the recommended protocol for neutral conditions, and adapting the gel electrophoresis methods for use in the Sub-Cell GT electrophoresis system (Bio-Rad, CA USA). Briefly, cells were collected from coverslips by treatment with 0.25% trypsin, pelleted and resuspended at 100,000 cells/ml in 1X DPBS (Ca2+ and Mg2+ free; Thermo Fisher Scientific) and verified to be greater than 95% viable by tryptan blue exclusion using an automated cell counter before continuing analysis. Aproximately 5,000 cells were embedded in low melting agarose, plated on slides and lysed overnight. The next day, electrophoresis was run at 30 Volts for 30 minutes in 1X TBE (National Diagnostics). Samples were fixed in 70% ethanol for 5 minutes, and slides were immersed in 1X TE buffer pH 8.0 (Ambion) with 1:10 of 10,000x SYBR green nucleic acid stain (Thermo Fisher Scientific). Fluorescent images were captured using a Leica DMI 400B inverted microscope for scoring.
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