Mouse competitive homing experiments were performed as described32 (link). In brief, BM cells from Fancb−/y or their male WT littermates (CD45.2+) were labeled with DiO dye at a density of 2 × 106 cells per ml at 37 °C for 30 min. In parallel, BM cells from BoyJ mice (CD45.1+) were labelled with DiD dye (1:200) at 37 °C for 30 min. DiO and DiD were purchased from Vybrant Multicolor Cell-Labelling Kit (Molecular Probes, V-22889). After labelling, dyes were washed off. The DiO-labelled CD45.2 and DiD-labelled CD45.1 cells were mixed at a 1:1 ratio and transplanted into lethally irradiated (11 Gy one day before transplantation) CD45.1 recipients (2.5 × 106 from each donor). Sixteen hours after transplant, the recipients were euthanized and the BM was analyzed by flow cytometry for both DiO/DiD and surface lineage markers (Gr1, Mac1, B220, CD3, Ter119, from Ebioscience) and c-Kit (2B8, BD Biosciences). The ratio between the percentages of DiO+ (donor) and DiD+ (competitor) cells within different cell populations was quantified.
Free full text: Click here
