Deltavision softworx

HCT116 cells and HeLa Kyoto cells expressing EGFP (enhanced green fluorescent protein)-α-tubulin, EGFP-CENP-A, and H2B-mCherry were grown in glass chambers (Thermo Fisher Scientific). Thirty minutes before imaging, the medium was changed to pre-warmed Leibovitz’s L-15 medium (Thermo Fisher Scientific) supplemented with 20% FBS and 20 mM HEPES, pH 7.0. Recording was performed in a temperature-controlled incubator at 37 °C. Z-series of five mCherry image sections in 3 μm increments were captured every 2 min. Images were obtained using an IX-71 inverted microscope (Olympus) controlled by DeltaVision softWoRx (GE Healthcare) using a 20 × 0.75 NA UPlanS Apochromat objective lens (Olympus). Deconvolution was performed when necessary, and image stacks were projected.

Immunolabeled membrane samples were loaded into a Chamlide chamber and immersed in a buffer containing 10% w/v glucose, 50 mM Tris pH 8, 10 mM NaCl, 10 mM cysteamine hydrochloride, 40 μg/mL catalase, 0.56 μg/mL glucose oxidase. Samples were imaged using the DeltaVision OMX V4 Monet Localization Microscopy system (GE Healthcare), with 642 nm laser TIRF illumination (60X oil immersion lens, Olympus, 1.49 NA) and a beam concentrator to assist fluorophores transition to the triplet (dark) state. A 405 nm laser was used to improve fluorophore cycling back to the ground state. Single events were captured over 10,000 frames. DeltaVision softWoRx (GE Healthcare) software was used to process data. Fluorescent events with a localization precision of 5–100 nm and persistence between 1–100 frames were included in the final reconstruction. Each event was fitted with a Gaussian distribution to localize the point of origin. Data was recolored in FIJI using the ‘Red Hot’ look-up table, where black, red, yellow and white indicate a gradient from low to high signal.

U2OS Flp-In T-REx parental cells or stably expressing siRNA-resistant venus-MBP-BRCA2 constructs were seeded in µ-Slide eight-well dishes (Ibidi). Alongside Ctrl or BRCA2 siRNA transfection as described above, cells were synchronized to S-phase with a single 24 h 2 mM thymidine block. Cells were released from the block, treated with 3 μM MMC for 1 h, and then allowed to recover for 8 h in normal growth medium. Cells were fixed and permeabilized by incubation in 4% formaldehyde for 10 min, 0.1% Triton-X-100 in PBS-T for 10 min, and 25 mM glycine for 20 min, followed by blocking in 3% BSA (Sigma) in PBS-T for 30 min. Cells were incubated with primary antibody, rabbit-anti-RAD51 (Bioacademia 70-001) 1:1000 in blocking solution, for 90 min, followed by washing in TBS-T and incubation with secondary antibody, AlexaFluor 546 nm Goat-anti-rabbit IgG (Life Technologies, A-11010) 1:1000 and 1 μg/mL DAPI, in blocking solution for 45 min. Finally, cells were washed in PBS-T and analysed on a Deltavision Elite microscope with a ×40 oil objective using DeltaVision SoftWoRx software (version 7.0.0., GE healthcare). Images were deconvoluted using DeltaVision SoftWoRx (version 5.0.0., GE healthcare), and Z stacks combined using the Quick projection function. The number of RAD51 foci in each nucleus was quantified using the polygon finder function. Graphs were constructed in PRISM.