Hematopoietic Stem Cell Analysis

MNCs from BM or FL were pre-incubated with biotin-conjugated anti-CD3e, anti-CD45R/B220, anti-Gr-1, anti-CD11b, and anti-Ter-119 antibodies, with anti-CD16/32 antibody to block the Fcγ receptors (CD11b antibody was excluded in FL MNCs)^{43 (link)–45 (link)}. The cells were then stained with streptavidin-fluorescein isothiocyanate (FITC) and anti-Sca-1-PE-Cy7, c-Kit-APC-Cy7, CD150-APC, and CD48-Pacific Blue antibodies. The frequencies of HPCs (Lin⁻/Sca-1⁻/c-kit⁺), LSK cells (Lin⁻/Sca-1⁺/c-kit⁺), and HSCs (CD150⁺/CD48⁻/LSK) were analyzed using an Aria II cell sorter. For each sample, approximately 5 × 10⁵ to 1 × 10⁶ cells were acquired and the data were analyzed using the BD FACSDiva 6.0 software (BD Biosciences) and the FlowJo software (FlowJo, Ashland, OR, USA) . The number of the different hematopoietic cell populations in each mouse was calculated by multiplying the total number of MNCs harvested from each mouse or embryo with the frequencies of each population of MNCs. The information for all antibodies used in the staining is provided in Supplementary Table 1.