Quantifying DNA Damage in Hematopoietic Stem Cells

Immunofluorescence staining for γH2AX was adapted from previously described^{16 (link)}. After co-culture in serum-free medium supplemented with SCF (20ng/ml) and TPO (10ng/ml) for 6 days, Lin⁻Sca-1⁺c-Kit⁺CD48⁻CD150⁺ HSCs were directly sorted on polylysine-coated slides (P0425–72AE; Sigma), 500–2000 cells per slide, incubated for 10 min, fixed with 4 % PFA for 10 min at room temperature, and permeabilised with 0.2 % Triton X-100 for 20 min at room temperature. Subsequently, the cells were blocked with 1% BSA/PBS overnight at 4 °C. Cells were then incubated with 0.125 μg (5μl of 25μg/ml) FITC-conjugated anti-phospho-H2A.X (Ser139) antibody (Clone: 2F3; Cat: 613404; BioLegend) for 2 h at 37°C. Primary antibody staining was followed by 3 washes with PBS and 10 min incubation with 0.2 % Hoechst 33342 (Sigma). All images were acquired using ZEISS AXIO examiner D1 microscope (Zeiss) with confocal scanner unit (Yokogawa), and reconstructed in three dimensions with Slide Book software (Intelligent Imaging Innovations). Image analysis was performed using the Slide Book software (Intelligent Imaging Innovations).