Chlorodeoxyuridine cldu

Nucleoside analogue administration was performed as previously described [4 (link)]. Briefly, 4-week-old mice received iododeoxyuridine (IdU; Sigma-Aldrich, Dorset, UK) in drinking water (1 mg/ml) for 3 weeks, followed by a 5-week wash-out period. Immediately after joint surface injury, mice received an injection of chlorodeoxyuridine (CldU; Sigma-Aldrich) subcutaneously (10 mg/ml) and then received CldU in their drinking water (1 mg/ml) for 7 days.

Camptothecin (Sigma-Aldrich) was dissolved in DMSO and a stock solution (10 mM) was prepared and stored at −20°C. Iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU) (Sigma-Aldrich) were dissolved in sterile DMEM to obtain stock solutions of 2.5 mM and 200 mM, respectively, and stored at −20°C. Mirin (Calbiochem), an inhibitor of MRE11 exonuclease activity, was used at 50 μM; the B02 compound (Selleck), an inhibitor of RAD51 activity, was used at 27 mM. The CDK inhibitor roscovitine (Selleck) was used at 20 μM.

Sub-confluent TREx BCBL-1-RTA cells were treated with 0.5 µg ml⁻¹ doxycycline for 10 h before chloro-deoxyuridine (CldU) (Sigma Aldrich), at a final concentration of 25 µM, was added to cell-culture media for 20 mins. Cells were pelleted and washed in media containing 250 µM iodo-deoxyuridine (IdU) (Sigma Aldrich) before being re-suspended in media containing 250 µM IdU for a total exposure to IdU of 20 mins. Cells were pelleted and re-suspended in ice cold PBS at a concentration of 5×10⁵ ml⁻¹. 2 µl of cell suspension was placed on glass slides, mixed with 7 µl of spreading buffer (200 mM Tris-HCl, pH 7.5, 50 mM EDTA, 0.5 % SDS) and spread across the glass by tilting at an angle. Slides were air dried and fixed in 3 : 1 methanol, acetic acid for 10 mins. For immunostaining of DNA fibres, DNA was denatured using 2.5 M HCL for 1 h, washed in PBS and submerged in blocking solution (1 % BSA, 1 % Tween 20 in PBS) for 30 mins. DNA immunostaining was carried out by submersion in rat anti-BrdU (clone BU1/75, ICR1; Abcam, ab6326), and mouse anti-BrdU (clone B44; BD Biosciences, 347583) in blocking buffer for 1 h. Following three washes in PBS-Tween followed by incubation with anti-rat Alexa Fluor 555 and anti-mouse Alexa Fluor 488 (Thermo Fisher Scientific). DNA fibres were visualised by immunofluorescence microscopy using a Leica DM6000B epifluorescence microscope.