Supplemental Information
Comet assay
Slide coating with agarose
Mix 1.5g agarose (Sigma) with 100ml PBS and melt (3x1minute) in a microwave. Mix and
transfer to a water bath at 600C. Dip microscope slides into the agarose, wipe the back and dry
overnight at room temperature. Store slides at 40C.
Stock Solutions
Lysis buffer base
2.5M NaCl 146.1g
100mM NaEDTA 37.2g
10mM Tris Base 1.2g
Add 890ml distilled water and adjust to pH 10 with 7g NaOH pellets store at 40C
10x Fpg enzyme buffer
400mM Hepes 19.1g
1M KCl 14.9g
5mM EDTA 372mg
2mg/ml BSA 400mg
Distilled water 200ml
Adjust pH to 8.0 with 5M KOH prepare 25ml aliquots and store at -200C
Neutralisation Buffer
Tris Base 48.5g
Distilled water 900ml
Adjust to pH 7.5 with HCl and make up to 1l with distilled water
Electrophoresis Buffer
NaOH 24g
200mM EDTA 745mg
Distilled water 2l
Adjust pH to > 13.0 and store at 40C
Procedure
Day 1 seed cells
Day 2 add treatments include a positive control consisting of 60µM hydrogen peroxide and
incubate for the required time.
Prepare working lysis buffer
133.5ml lysis buffer base
15ml DMSO
1.5ml Triton-X
Preparation of low melting point agarose (LMP agarose)
50mg LMP agarose
10ml PBS
Microwave 2x 1 minute and keep in a water bath at 370C
Remove the cells from the plates (after the required incubation period) and suspend in 1ml RPMI
medium plus 10% FCS. Keep on ice.
Preparation of slides
Label slides, two are required for each treatment (one minus FPG and one plus FPG). In an
eppendorf tube add 20µl of cell suspension and 240µl LMP agarose and mix. Pipette 125µl of
this mixture (quickly) on to agarose coated slides and cover with a coverslip. Store slides on ice
for 10 minutes. Remove the coverslips and place the slides in coplin jars containing lysis buffer.
Cover with foil and store overnight at 40C.
Day 3
Prepare 1x FPG enzyme buffer
25ml 10x FPG enzyme buffer
225ml distilled water
Dilute FGP enzyme in the FPG buffer (the exact dilution needs to be determined by running a
series of control slides) 1:30 and keep on ice.
Wash slides 3x5 minutes in FPG buffer, dry for 1 minute and place all slides on ice. For the FPG
+ slides add 100µl prepared FPG enzyme (1 unit in 50µl FPG buffer); for the FPG – slides add
100µl FPG buffer. Cover with coverslips and incubate at 370C for 30 minutes. Place slides in an
electrophoresis tank (cooled to 40C), slides should all face the same direction and cover with
electrophoresis buffer. Incubate for 20 minutes. Apply current, 24v and 270mA (protected from
light) for 20 minutes. The electrophoresis volts and amps can be adjusted by adding or removing
electrophoresis buffer 25ml at a time. Remove slides and place in neutralisation buffer for 15
minutes. Dry the slides for 15 minutes. Slides may be stored and stained later by dipping in
100% ethanol and allowing to dry.
Staining
Dilute GelRed (Biotium Cat No 41003-0.5ml) 1:10000 in PBS
Add 40µl diluted stain per slide and cover with a coverslip. Store in the dark. Examine by
fluorescence microscopy and score using Comet software.