Chromosome metaphase spreads

For genome instability analysis, B cells isolated from animals between 8–12 weeks of age were harvested after 3 days in culture stimulated to undergo class switching to IgG1. Metaphase spreads were prepared and processed for FISH analysis as previously described ^9–12. PARP inhibitor Olaparib (2µM, AZD2281, Selleckchem) was added to cells stimulated ex vivo for 16 h and Colcemid (100 ng/ml, Roche) added 1 h before preparation of metaphase spreads, and imaging as described below using a high content microscope. Experiments were performed with the investigator blinded to the group allocation. An assistant labeled the slides and/or dissected spleen/cultured cells before analysis by the investigator, and the data were subsequently related to the identity of the specimens. A total of 165 (WT) and 189 (SCAI^−/−) metaphase spreads from DMSO-treated cells and 452 (WT) and 453 (SCAI^−/−) spreads from PARPi-treated cells were analyzed, across multiple mice, and detailed in the Table S3. Spermatocyte metaphase spreads were prepared as previously described ¹³, except a 2.9% isotonic sodium citrate dihydrate solution was used and the slides were stained with Giemsa. Spermatocyte metaphase spread images were acquired on a Zeiss Axio Scope.A1 LED with a Plan-Apochromat 100X/1.4 oil immersion objective.