Asymmetric Division in HSCs

Numb/α-tubulin (Numb: ab4147, Tubulin: ab7291, Abcam, Cambridge, UK) staining in FACS-purified LSKs or LT/ST-HSCs from chimeric mice, 5-FU-treated BL/6 and Ltbr^−/− mice (8 days after treatment) or CML mice was performed as follows: cells were fixed with 4% paraformaldehyde, followed by permeabilization with 1× wash buffer (Dako wash, Agilent Technologies, California, USA) and blocking with 10% normal goat serum (Invitrogen, California, USA) in Dako wash. After overnight incubation at 4 °C with the primary rabbit α-Numb antibody or tubulin in Dako diluent, cells were incubated with the secondary antibody (donkey-anti-goat, ab175474; goat-anti-mouse, ab150115) for 1 h at room temperature^{63 (link)}. DAPI was used to stain for DNA. Asymmetric cell division was determined by an increase in Numb intensity of 1.8-fold in one of the daughter cells^{11 (link)}. Cells were acquired using an ImageStreamX MkII imaging flow cytometer (Merck, Darmstadt, Germany). Cells were analyzed using INSPIRE and IDEAS Software^{63 (link)}.