Flow cytometric analysis

Transduction efficiency of the transferred gene was evaluated by flow cytometric analysis in WBCs from wild type and SOD1(G93A) mice at 3 and 6 weeks after BMT because it takes approximately three to four weeks for the stable engraftment of bone marrow cells after transplantation. WBCs in 200 µl blood were collected from each mouse by cutting the tip of tail, and centrifuged at 100×g for 5 min at 4 °C. After the pellet of blood cells was resuspended with 1 ml lysis buffer (154 mM NH₄Cl, 2 mM NaHCO₃ and 0.1 mM EDTA₂Na), that was centrifuged at 100×g for 5 min at 4 °C. Then, 1 ml PBS (−) was added to the cell pellet, followed by centrifugation at 100×g for 5 min at 4 °C again. After 500 µl PBS (−) was added to the cell pellet, for the cells were analyzed by flow cytometry. Emission at 530 was induced by excitation of a 488 nm laser. The gate of the fluorescence threshold was determined using WBCs from C57BL/6 mice after BMT from GFP or wild type mice as positive and negative controls, respectively. A FACS Canto II with FACS DIVA software (BD Biosciences) was used for data collection and analysis.