Fig. 4. Experimental design of in vitro clonogenic and in vivo long-term reconstitution assays. Single-cell suspensions were prepared from the AGM region (E11.5 and E12.5) and liver (E11.5, E12.5 and E14.5) of control C57Bl6 and Mpl^-/- embryos (expressing the Ly5.2 allele of CD45), and analyzed in vitro in clonogenic assays, or in vivo for long-term reconstitution (LTR) of lethally irradiated C57Bl6 mice expressing the Ly5.1 allele of CD45. At E12.5, single-cell suspensions of yolk sac or peripheral blood were also tested. The engraftment and repopulating activity of the injected cells were assessed at different times after injection (5, 10 and 20 weeks) by FACS analysis on the basis of the detection of Ly5.2 in the peripheral blood (minimal level of reconstitution considered for positive reconstitution was 5%). Twenty weeks after injection, LTR ability (LTRA) was evaluated (FACS analysis) in positive mice by the percentage of Ly5.2⁺ cells present in the bone marrow (BM). The HSC content was thereafter determined by secondary transplantation of bone marrow cell suspensions from primary recipients into lethally irradiated B6-Ly5.1 mice.