3T3 or HeLa cells (or others)
Hydrogen peroxide 30% (H1009, Sigma)
Methylmethane sulfonate (MMS) (Aldrich)
Primary antibodies : 10H monoclonal anti-poly(ADP-ribose) antibody or polyclonal anti-poly(ADP-ribose) antibody (LP96-10, Alexis Corp.), anti-XRCC1 (Alexis Corp.)
Secondary antibodies : goat anti-mouse or goat anti-rabbit antibodies conjugated to Alexa Fluor™ 568 or Alexa Fluor™ 488 (Molecular Probes)
DAPI stain stock solution : 0.25 mg/ml in 50% glycerol-PBS 1x
Mowiol (4-88 Hoechst)
DABCO anti-fading reagent (Sigma)

Solutions :

PBS buffer
PBS + 0.1% Tween (v/v) + 0.1% non fat dry milk
DNA staining stock solution : 0.25 mg/ml DAPI in PBS, 50% glycerol (store at -20°C)
Mounting solution : add 2.4 g of Mowiol to 4.8ml of 100% glycerol, stir and add 6 ml H₂0. Stir at room temperature for several hours ; add 12 ml 0.2M Tris-HCl pH 8.5 and incubate at 50°C until dissolved. Add 0.45 g DABCO antifading reagent at 4°C (2.5% DABCO (w/v) final concentration). Aliquote and store at -20°C

Protocol :

1. Whole cell damage : Expose cultured cells (i.e. 3T3 or HeLa) to either 0.1-1 mM hydrogen peroxide for 10 min at 37°C or 1 mM MMS for 30 min at 37°C.

2. Fixation : Wash damaged cells three times with ice cold PBS, fix cells with methanol/acetone (1:1, v/v) for 10 min at 4 °C and wash again 3 times for 5 min with ice cold PBS supplemented with 0.1% Tween (v/v).

3. Primary antibodies : Cells are incubated overnight at 4°C with the monoclonal anti-poly(ADP-ribose) antibody (10H) (1:200 dilution) or with the polyclonal anti-poly(ADP-ribose) antibody (1:2,000) diluted in 1x PBS, 0.1% Tween, 0.1% non fat dry milk.

4. Secondary antibodies : After washing 3 times for 5 min in PBS, 0.1% Tween (v/v), the cells are incubated for 2 hr at room temperature (or for 4 hr at +4°C) with a 1:1,500 dilution of goat anti-mouse or goat-anti rabbit fluorophore-conjugated antibodies in 1x PBS, 0.1% Tween (v/v), 0.1% non fat dry milk.

5. DNA staining : The cells are washed once for 5 min in 1x PBS, stained for 10 min at room temperature with 1:20,000 dilution in PBS of the DNA staining stock solution, and washed twice again in PBS.

6. Mounting : The coverslides are deeped in water and mounted using 25 µl of Mowiol containing DABCO antifading reagent.

Poly(ADP-ribose) synthesis and XRCC1 recruitment at laser induced DNA strand breaks :

1. Cells are grown onto 55 µm square size CELLocate coverslips (Eppendorf, Hamburg, Germany).

2. The cells are incubated with 10 µg/ml Hoechst dye 33258 in DMEM medium for 10 min at 37°C under 5% CO₂.

3. Laser microirradiation is performed with a Leica DM LMD microscope (Leica Microsystems, Wetzlar Germany) fitted with a 337.1 nm laser focused through a 40x or a 63x objective. The peak performance is 7.2 mW at the maximum pulse repeat rate of 30 Hz. The laser is guided over cell nuclei using a joystick to draw the laser path. The irradiation is performed using the following features of the laser: aperture 2, power 15, speed 5, off-set 30. Cell positioning on CELLocate coverslips is recorded by phase contrast imaging of irradiated cells.

4. After irradiation, cells are fixed and immunostained as described above.

5. DNA strand breaks can be detected by deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) (ApoAlert DNA fragmentation assay kit, Clontech).