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

Summary CD4+CD25+ T regulatory cells (Tregs) play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. Here we explore if Tregs also play a role in controlling the immediate hypersensitivity response of mast cells (MCs). Tregs directly inhibit the FcεRI-dependent degranulation of MCs through cell-cell contact involving OX40-OX40L interactions between Tregs and MCs, respectively. MCs show increased cAMP levels and reduced Ca2+ influx, independent of PLC-γ2 or Ca2+ release from intracellular stores. Antagonism of cAMP in MCs reverses the inhibitory effects of Tregs restoring normal Ca2+ responses and degranulation. Importantly, the in vivo depletion or inactivation of Tregs causes enhancement of the anaphylactic response. The demonstrated cross-talk between Tregs and MCs defines a previously unrecognized mechanism controlling MCs degranulation. Loss of this interaction may contribute to the severity of allergic responses.