These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer‐reviewed by leading experts in the field, making this an essential research companion.

International audienceThe classical model of hematopoiesis established in the mouse postulates that lymphoid cells originate from a founder population of common lymphoid progenitors. Here, using a modeling approach in humanized mice, we showed that human lymphoid development stemmed from distinct populations of CD127(-) and CD127(+) early lymphoid progenitors (ELPs). Combining molecular analyses with in vitro and in vivo functional assays, we demonstrated that CD127(-) and CD127(+) ELPs emerged independently from lympho-mono-dendritic progenitors, responded differently to Notch1 signals, underwent divergent modes of lineage restriction, and displayed both common and specific differentiation potentials. Whereas CD127(-) ELPs comprised precursors of T cells, marginal zone B cells, and natural killer (NK) and innate lymphoid cells (ILCs), CD127(+) ELPs supported production of all NK cell, ILC, and B cell populations but lacked T potential. On the basis of these results, we propose a "two-family" model of human lymphoid development that differs from the prevailing model of hematopoiesis

Key Points• Comparative global gene expression analysis of primary murine primitive, fetal definitive, and adult definitive erythroid precursors.• Primitive erythroblasts contain and accumulate high ROS levels and uniquely express the H2O2 transporting aquaporins 3 and 8.Erythroid ontogeny is characterized by overlapping waves of primitive and definitive erythroid lineages that share many morphologic features during terminal maturation but have marked differences in cell size and globin expression. In the present study, we compared global gene expression in primitive, fetal definitive, and adult definitive erythroid cells at morphologically equivalent stages of maturation purified from embryonic, fetal, and adult mice. Surprisingly, most transcriptional complexity in erythroid precursors is already present by the proerythroblast stage. Transcript levels are markedly modulated during terminal erythroid maturation, but housekeeping genes are not preferentially lost. Although primitive and definitive erythroid lineages share a large set of nonhousekeeping genes, annotation of lineage-restricted genes shows that alternate gene usage occurs within shared functional categories, as exemplified by the selective expression of aquaporins 3 and 8 in primitive erythroblasts and aquaporins 1 and 9 in adult definitive erythroblasts. Consistent with the known functions of Aqp3 and Aqp8 as H 2 O 2 transporters, primitive, but not definitive, erythroblasts preferentially accumulate reactive oxygen species after exogenous H 2 O 2 exposure. We have created a user-friendly Web site (http:// www.cbil.upenn.edu/ErythronDB) to make these global expression data readily accessible and amenable to complex search strategies by the scientific community. (Blood. 2013;121(6):e5-e13) IntroductionRBCs constitute an estimated 1 in 4 cells in the body and are necessary for tissue oxygen delivery. In the adult, RBCs are produced primarily in the BM where lineage-committed progenitors give rise to morphologically identifiable precursors. Erythroid precursors physically associate with macrophages and undergo several maturational cell divisions characterized by a progressive decrease in cell size, nuclear condensation, hemoglobin accumulation, and loss of RNA content. 1 These physical changes have been used to classify erythroid precursors into proerythroblast, basophilic, polychromatophilic, and orthochromatic erythroblast maturational stages. In mammals, orthochromatic erythroblasts enucleate to form reticulocytes that ultimately enter the circulation and complete their maturation.Erythroid cells are a critical component of the cardiovascular network, which constitutes the first functional organ system in the mammalian embryo. 2 "Primitive" erythroid cells first emerge in yolk sac blood islands. 3 We previously determined that primitive erythroid cells originate from a transient wave of committed progenitors in the yolk sac and mature as a semisynchronous cohort in the bloodstream, undergoing morphologic changes similar to those observed in defini...

Mammals have 2 distinct erythroid lineages. The primitive erythroid lineage originates in the yolk sac and generates a cohort of large erythroblasts that terminally differentiate in the bloodstream. The definitive erythroid lineage generates smaller enucleated erythrocytes that become the predominant cell in fetal and postnatal circulation. These lineages also have distinct globin expression patterns. Our studies in primary murine primitive erythroid cells indicate that ␤H1 is the predominant ␤-globin transcript in the early yolk sac. Thus, unlike the human, murine ␤-globin genes are not up-regulated in the order of their chromosomal arrangement. As primitive erythroblasts mature from proerythroblasts to reticulocytes, they undergo a ␤H1-to ⑀y-globin switch, up-regulate adult ␤1-and ␤2-globins, and down-regulate -globin. These changes in transcript levels correlate with changes in RNA polymerase II density at their promoters and transcribed regions. Furthermore, the ⑀y-and ␤H1-globin genes in primitive erythroblasts reside within a single large hyperacetylated domain. These data suggest that this "maturational" ␤H1-to ⑀y-globin switch is dynamically regulated at the transcriptional level. Globin switching during ontogeny is due not only to the sequential appearance of primitive and definitive lineages but also to changes in globin expression as primitive erythroblasts mature in the bloodstream. IntroductionThe first blood cells to circulate during mammalian embryogenesis consist of large primitive red cells. In the mouse embryo, primitive erythroid cells are generated from a transient wave of committed progenitors found in the yolk sac between embryonic day (E) 7.25 and E9.0 of gestation. 1,2 Primitive proerythroblasts begin to emerge from blood islands beginning at E8.25, and enter the newly forming bloodstream, 3 where they undergo terminal maturation. They transition as a semisynchronous cohort of cells from proerythroblasts to enucleated erythrocytes progressively undergoing a loss of proliferative capacity, a decrease in cell size, accumulation of hemoglobin, and nuclear condensation. [4][5][6] Ultimately, they enucleate by E16.5 to become primitive erythrocytes that can circulate for several days after birth. 7 After E11.5, primitive red cells are joined by increasing numbers of smaller definitive erythrocytes that are released from the fetal liver after enucleating. Over the ensuing 5 days, definitive erythrocytes become the predominant cell type in the fetal circulation and ultimately become the exclusive red-cell lineage in the adult.The sine qua non of red cells is their accumulation of large amounts of hemoglobin composed of globin tetramers encoded by the ␣-and ␤-globin gene loci. Examination of globin expression in circulating human and mouse blood cells indicated that 5Ј members of both globin loci are expressed in the embryo, while 3Ј members are expressed in the adult, leading to the concept of globin switching during ontogeny. 8 Since this switch in globin expression coincided temporally with t...

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