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

The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity and the extent of background staining. As previously reported, monochlorobimane was highly specific for GSH in rodent cells but failed to label human cells adequately because of its low affinity for human glutathione S-transferases. Higher concentrations of monochlorobimane achieved more complete labelling of the human cellular GSH pool but gave increased background fluorescence due to non-GSH binding. The analogue monobromobimane, which binds nonenzymatically to sulphydryls, reacted more readily with GSH than with protein sulphydryls and, provided that stain concentration and incubation time were controlled, gave reproducible staining of human cells with -20% of total fluorescence due to background staining. Of the currently available stains for measuring GSH in human cells, monobromobimane is the agent of choice. Mercury orange also binds more readily to GSH than to protein, giving a degree of specificity, and it has the additional advantage of being excited at 488 nm. However, the reproducibility of staining with mercury orange was less consistent than that using monobromobimane, and a higher background fluorescence was seen. Two additional stains, o-phthaldialdehyde and chloromethyl fluorescein, could also be used to label cellular GSH, but both gave an unacceptably high level of background staining. It is recommended that flow cytometric GSH assays should routinely include a sample of cells that have been depleted of GSH in order to determine the extent of background labelling. 0 1994 Wiley-Liss, Inc.

Cancer cells frequently show abnormal signaling via the mitogen activated protein kinase (MAP kinase) pathway due to increased activity of surface receptors for growth factors, or as a result of ras mutations. The development of potent anti-cancer agents that target this pathway prompts the need for analytical methods that allow pharmacodynamic monitoring of drug effects in patients during early phase clinical trial. We describe such a method, based on the activation of T-lymphocytes in undiluted peripheral blood using phorbol myristate acetate (PMA). Following rapid hypotonic lysis and formaldehyde fixation, activation of the MAP kinase pathway can then be demonstrated using phospho-specific antibodies that recognize the activated mediators MEK or ERK, followed by surface labeling with anti-CD3 to identify T-lymphocytes. This method was used to investigate the effects of a MEK inhibitor, U0126, and a new raf kinase inhibitor BAY 37-9751 in blood samples from normal donors. Dose-dependent inhibition of pERK activation was demonstrated for both agents. Furthermore, differential effects on pMEK activation allowed the molecular targets of the two inhibitors to be distinguished. In addition to monitoring drug effects in patients during treatment with inhibitors of the MAP kinase pathway, the general methodology described in this paper has the potential for wide application to the study of signal transduction at the single cell level using flow cytometry. Cytometry (Comm. Clin. Cytometry) 46:72-78, 2001.