CometAssay Electrophoresis System II

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 50,000 cells per well in a 96-well plate		Comets can be imaged by using the LionHeart FX automated microscope (Biotek) and the Gen5 Image Prime software to construct image montages that were analyzed using TriTek CometScore 2.0.0.38.

Upstream tips
Seed 50,000 cells per well in a 96-well plate
Downstream tips
Comets can be imaged by using the LionHeart FX automated microscope (Biotek) and the Gen5 Image Prime software to construct image montages that were analyzed using TriTek CometScore 2.0.0.38.

Publication protocol

Alkaline single-cell electrophoresis was performed using the CometAssay Reagent Kit (Trevigen catalog no. # 4250–050) in accordance with the manufacturer's instructions. Briefly, cells were plated at a density of 50,000 cells per well in a 96-well plate, incubated for 24 hours, then treated with 0.2% DMSO, 1 μmol/L TAL, 1 μmol/L SN-38, or a combination of 1 μmol/L TAL and 1 μmol/L SN-38. After being treated for 2.5 hours, the cells were trypsinized then washed with ice-cold 1× PBS. They were then combined with low-melting-point agarose (LMAgarose, Trevigen catalog no. # 4250–500–02) at a ratio of 1:10 [volume for volume (v/v)] and immediately plated onto CometSlides (Trevigen catalog no. # 4253–096–03). The slides were placed at 4°C in the dark for 30 minutes to improve cell adherence then immersed in lysis solution for 60 minutes. The slides were then drained and incubated with Alkaline Unwinding Solution for 20 minutes at room temperature. Electrophoresis was then performed using the CometAssay Electrophoresis System II unit (Trevigen catalog no. # 4250–050-ES) with 21 volts being applied for 30 minutes for ES8 and ES8-SLFN11-KO cells and for 45 minutes for CHLA-258. Samples were dried for 30 minutes at 37°C and washed with dH2O × 2 and 70% ethanol. Samples were then dried for an additional 30 minutes at 37°C then stained with SYBR Gold solution for 30 minutes. Comets were imaged by using the LionHeart FX automated microscope (Biotek) and the Gen5 Image Prime software to construct image montages that were analyzed using TriTek CometScore 2.0.0.38.

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