CometAssay Electrophoresis System II

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Cell suspension at a concentration of 10^5cells/ml was mixed in a 1:1 ratio withTrevigen LMAgarose (#4250-050-02) at 37◦C.		The images of the comets were analyzed using CellProfiler software (version 2.1.1 rev 6c2d896).

Upstream tips
Cell suspension at a concentration of 10^5cells/ml was mixed in a 1:1 ratio withTrevigen LMAgarose (#4250-050-02) at 37◦C.
Downstream tips
The images of the comets were analyzed using CellProfiler software (version 2.1.1 rev 6c2d896).

Publication protocol

After treatment, cells were trypsinized with 0.25% trypsin for several minutes at 37◦C. The trypsin was inactivatedwith a 4-fold volume of DMEM. Cell suspension at a concentration of 10^5cells/ml was mixed in a 1:1 ratio withTrevigen LMAgarose (#4250-050-02) at 37◦C. The mixture was pipetted onto comet slides (Trevigen, #3950-300-02)that had been pre-coated with a 1% normal melting point agarose (Sigma) base layer. The drop containing the cells was covered with a glass coverslip and incubated at 4◦C for 5 min. After incubation, the coverslips were removed, and the slides were immersed in lysis solution (30 mM ethylene-diaminetetraacetic acid (EDTA), 0.5% sodium dodecyl sulphate (SDS) and 10 mM Tris–HCl, pH 8.0, supplemented with 500g/ml proteinase K) and incubated at 37◦Cfor1 h. After lysis, the slides were washed three times for 5min in PBS and incubated in 1×TBE (Tris-Borate-EDTAbuffer) for 20 min at 4◦C. Electrophoresis was performed in a Trevigen electrophoresis system (#4250-050-ES) for 10min at 4◦Cand1V/cm in 1×TBE. The comets were counterstained with SYBR Green for 1 h (1:3000; Thermo Sci-entific, #S7563). The comets were visualized at four magni-fication using an inverted Nikon Eclipse TiE fluorescence microscope equipped with a Nikon Intensilight C-HGFIlight source (objective: Nikon Plan Fluor 4/0.13; camera:DS-Qi2). The images of the comets were analyzed usingCellProfiler software (version 2.1.1 rev 6c2d896).

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