Computational protocol: Human RECQ1 promotes restart of replication forks reversed by DNA topoisomerase I inhibition

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Protocol publication

[…] U-2 OS cells were grown on coverslips, fixed in 3.7% paraformaldehyde, permeabilized in 0.5% Triton X-100 and blocked in 3% BSA. Coverslips were then stained with rabbit polyclonal anti-53BP1 (Novus Biologicals, NB100-304) and mouse monoclonal anti-γH2AX (Millipore, 05-636), and detected by appropriate Alexa 488- and Alexa 594-conjugated secondary antibodies. Toto3 Iodide (Life Technologies, T3604) was used as a nuclear counterstain. Cells were imaged using a Zeiss LSM 510 Meta confocal microscope. Images were acquired using the LSM 5 software. Foci were counted with ImageJ “Analyze particles” function and “JACoP” plugin was used to calculate colocalization. The average number of foci was obtained from 3 independent experiments analyzing at least 35 cells per sample. […]

Pipeline specifications

Software tools ImageJ, JACoP
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Homo sapiens
Chemicals Poly Adenosine Diphosphate Ribose