Slide Coating with Agarose

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.