Anti ckit apc

For evaluation of MLL-AF9-transduced leukemia development, either peripheral blood or BM cells were stained with anti-Mac-1-APC, anti-Gr-1-PE, anti-CD3-APC, anti-B220-PE or anti-c-Kit-PE monoclonal antibodies (eBioscience) for the analyses of the myeloid and lymphoid lineages, differentiation, and cell frequencies of Mac-1 + c-Kit + LICs. For examination of Lin À CD127 À Sca-1 À c-Kit + CD16/32 + CD34 + L-GMP cells, BM leukemia cells were stained with biotinylated anti-CD3, anti-CD8, anti-B220, anti-Gr-1, anti-Ter119 and anti-CD127 antibody followed by staining with streptavidin-PE/Cy5.5, anti-Sca-1-PE/CY7, anti-c-Kit-APC, anti-CD16/ 32-eFluo450 and anti-CD34-PE (eBioscience). To determine the ratios of SoNar fluorescence, cells were excited at 405 nm/488 nm and acquisited at 525 nm (for SoNar sensor), or excited at 405 nm/561 nm and acquisited at 525 nm/610 nm (for SoNar-mCherry sensor), respectively. The mean fluorescence intensities and ratio (405/561) histograms were analyzed for the calculation of SoNar's signal by using FlowJo software v10. For the evaluation of homing ability, CXCR4 antibody (eBioscience) was used. The cell cycle status of SoNar-high and low AML cells were measured by staining with Hoechst 33342/Pyronin Y as previously described (Zheng et al., 2011) .