Materials and Reagents

1. Superfrost microscopy slides (ThermoScientic, Menzel-Gläser, Superfrost Plus, catalog number: J1800AMNZ), store at room temperature. The use of charged slides is necessary to ensure proper adhesion of agarose to the slides and to minimize sample loss during handling

2. Rectangular coverslips (Coverslips 22 × 50 mm; ThermoScientic, Menzel-Gläser, catalog number: MA062210), store at room temperature

3. 24-well cell culture plates (TPP, catalog number: 92024), store at room temperature

4. 15 ml Falcon tubes (Greiner, catalog number: 188271 Zentrifugenröhrchen 15 ml, Producer: Huberlab AG, catalog number: 7.188 271), store at room temperature

5. Trypsin 10× stock solution (Gibco, catalog number: 15090-046), store stock solution at -20°C. Dilute 1:10 in 1× PBS for a working solution; this is stable at 4°C for several weeks

6. Potassium chloride (KCl) (Merck, catalog number: 1.04936.1000), store at room temperature

7. Normal melting point agarose (Standard Agarose-Type LE; BioConcept, catalog number: 7-01P02-R), store at room temperature

8. Low melting point agarose (Low Melt Agarose 100 g; Bio & Sell, catalog number: BS20.47.100), store at room temperature

9. Na₂HPO₄·7H₂O (Sigma-Aldrich, catalog number: S9390-1Kg), store at room temperature

10. KH₂PO₄ (Sigma, catalog number: 602187), store at room temperature

11. NaCl (Sigma, catalog number: 71380-1KG), store at room temperature

12. EDTA disodium salt dihydrate (C₁₀H₁₄N₂Na₂O₈·2H₂O) (Sigma-Aldrich, catalog number: 03685-1KG), store at room temperature

13. Tris base (Sigma, Life science, catalog number: T1503-500G), store at room temperature

14. Sodium hydroxide pellets (NaOH) (Merck, catalog number: 1.06498.100), store at room temperature

15. DMSO (Sigma-Aldrich, catalog number: D5879-1L), store at room temperature

16. Triton X-100 (MP-biomedicals/LLC, catalog number: 807426), store at room temperature

17. SYBR Gold nucleic acid gel stain (Life Technologies, catalog number: S11494), aliquot and store at -20°C protected from light

18. 30% hydrogen peroxide (H₂O₂) (Roth, catalog number: 8070.2), store at 4°C

19. Methyl-methanesulfonate (MMS) (Sigma, catalog number: 129925), make 10 M solution in sterile dH₂O, aliquot, and store at -20°C

20. Lysis buffer for the alkaline comet assay (see Recipes)

21. Electrophoresis buffer for the alkaline comet assay (see Recipes)

22. Comet staining solution (see Recipes)

23. PBS (phosphate-buffered saline) (see Recipes)

Equipment

1. Trevigen CometAssay ESII apparatus (Trevigen, catalog number: 4250-050-ES)

2. Fluorescence microscope capable of excitation at 470-530 nm to image SYBR-Gold (excitation maximum around 495 nm, emission approx. 537 nm)

3. Water bath capable of maintaining a constant temperature of 37°C and large enough to hold a 250-ml glass bottle

4. Big styrofoam box or similar, large enough to hold a glass plate for 10-12 comet slides on ice

5. Small mechanic’s level

6. Coplin jars capable of holding 5 or 10 slides

7. ‘Humid chamber’ consisting of a plastic box with a lid, large enough to hold 10-12 slides on a wet paper towel and be placed in an incubator at 37°C

Software

1. Fiji image processing package (https://imagej.net/Fiji)

2. OpenComet plugin for Fiji (http://www.cometbio.org/) (Gyori et al., 2014)

Procedure