U-2 OS cells were grown on coverslips and sensitized to laser induced DSB formation using 5-Bromo-2-deoxyuridine (B9285-50MG, Sigma-Aldrich) for 24 h. GFP-SLF2 expression vectors were transiently transfected 24 h prior and GFP-SMC5 stable expressing cells were used for micro-irradiation. Laser micro-irradiation induced DSB formation was performed as previously described70 (link) with 1 h allowed for recovery. Cells were pre-extracted using CSK buffer (100 mM NaCl, 10 mM HEPES, 3 mM MgCl2, 300 mM Sucrose, 0.25% Triton-X-100, 1 mM PMSF) prior to fixation in formalin buffer (AMPQ43182, VWR) for 15 mins at room temperature (RT).
Fixed coverslips were blocked with 5% Bovine Serum Albumin (A7906, Sigma-Aldrich) for 1 h prior to staining with anti-γ-H2AX (Ser139) (1:1000, 05-636, Merck) and anti-GFP (1:500, PABG1, Chromotek) overnight at 4 °C. After PBS washes cells were stained with Alexa Fluor secondary antibodies and 4’,6-Diamidino-2-Phenylindole (DAPI, D1306, Molecular Probes) for 30 min at RT. After further washing, coverslips were dried completely and mounted for imaging using Mowiol (81381, Sigma-Aldrich).
Fixed coverslips were blocked with 5% Bovine Serum Albumin (A7906, Sigma-Aldrich) for 1 h prior to staining with anti-γ-H2AX (Ser139) (1:1000, 05-636, Merck) and anti-GFP (1:500, PABG1, Chromotek) overnight at 4 °C. After PBS washes cells were stained with Alexa Fluor secondary antibodies and 4’,6-Diamidino-2-Phenylindole (DAPI, D1306, Molecular Probes) for 30 min at RT. After further washing, coverslips were dried completely and mounted for imaging using Mowiol (81381, Sigma-Aldrich).
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