Vista green dna dye

Genotoxic effect of 20b (at the concentration representing its IC_50Tg against RH-GFP strain) was carried out using a single cell gel electrophoresis technique (comet assay) based on OxiSelect Comet Assay Kit (Cell Biolabs, Inc., San Diego, CA). All the procedures performed were in line with the manufacturer’s protocol. Human Hs27 cells, previously exposed for 24 h to the compound, were combined with liquefied low melting Agarose at 37 °C and transferred onto the OxiSelect comet slides. After storing for 15 min at 4 °C, the slides were immersed in a pre-chilled Lysis Buffer (for 45 min at 4 °C) and Alkaline Solution (for 30 min at 4 °C). Afterwards, the Alkaline Solution was replaced with pre-chilled TBE electrophoresis solution. After immersing for 5 min, the slides were put into the horizontal electrophoresis chamber and covered with TBE electrophoresis buffer. The electrophoresis was run for 15 min at 1 volt/cm. Finally, DNA was stained with Vista Green DNA Dye for 15 min at room temperature and observed under fluorescence microscope (Olympus BX63, Tokyo, Japan). The images of cells were captured using XM10 digital camera (Olympus). The DNA damage was measured quantitatively using OpenComet software. At least 50 randomly selected images were used in each analysis.

The genotoxic potential of Aloe arborescens extracts was evaluated using a single-cell gel electrophoresis technique based on OxiSelect Comet Assay Kit (Cell Biolabs, Inc., San Diego, CA, USA). All the procedures performed were in line with the manufacturer’s protocol. Human Hs27 cells, previously exposed for 24 h to the Aloe vera extract, were combined with liquefied low melting agarose at 37 °C and transferred onto the OxiSelect comet slides. After storing for 15 min at 4 °C, the slides were immersed in a pre-chilled Lysis Buffer (45 min, 4 °C) and Alkaline Solution (for 30 min at 4 °C). Afterward, the Alkaline Solution was replaced with a pre-chilled TBE (Tris/borate/EDTA) electrophoresis solution. After immersing for 5 min, the slides were put into the horizontal electrophoresis chamber and covered with TBE electrophoresis buffer. The electrophoresis was run for 15 min at 1 V/cm. Finally, DNA was stained with Vista Green DNA Dye for 15 min at room temperature and observed under a fluorescence microscope (Olympus BX63). The images of cells were captured using XM10 digital camera (Olympus). The DNA damage was measured quantitatively using ImageJ software (NIH, Bethesda, MD, USA). Etoposide was used as the positive control. At least 50 randomly selected images were used in each analysis.