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 paramagnetic resonance spectra of two spin-labels, 2,2,6,6-tetramethylpiperadinyl-1-oxy and a head-group spin-labeled phosphatidylethanolamine (L-alpha-dipalmitoylphosphatidyl-N-ethanolamine), have been used to study solid-liquid and liquid-liquid phase separations in binary mixtures of dimyristoylphosphatidylcholine and cholesterol. A quantitative analysis of these resonance spectra supports the view that at temperatures below theta m, the chain-melting temperature of the phospholipid, and at cholesterol mole fractions Xc less than 0.2, these mixtures consist of two phases, a solid phase of essentially pure dimyristoylphosphatidylcholine and a fluid phase having a mole fraction of cholesterol equal to 0.2. The spin-label data also provide evidence for fluid-fluid immiscibility in the bilayer membrane at temperatures above the chain melting transition temperature of dimyristoylphosphatidylcholine.

A flow cytometric assay was developed to detect rare cancer cells in blood and bone marrow. Multiple markers, each identified by a separate color of immunofluorescence (yellow and two shades of red), are used to reliably identify the cancer cells. Blood or bone marrow cells, which are not of interest but interfere in detecting the cancer cells, are identified by a panel of immunofluorescence markers, each of which has the same color (green). Thus, the rare cancer cells of interest are yellow and two different shades of red but not green. The requirement that the rare cancer cell be simultaneously positive for three separate colors (the specific markers) and negative for a fourth color (the exclusion color) allowed detection of as few as one cancer cell in 107 nucleated blood cells (a frequency of 10-7). To test this rare-event assay prior to clinical studies, a model study was performed in which the clinical sample was simulated by mixing small numbers of cells from the breast carcinoma line BT-20 with peripheral blood mononuclear cells. We detected statistically significant numbers of BT-20 cells at mixing frequencies of 10-5, 10-6, and 10-7. In control samples, no target events were observed when more than 108 cells were analyzed. For additional confirmation that the BT-20 cells in the model study were correctly identified and counted, the BT-20 cells (and only were covalently stained with a fifth fluorescent dye, 7-amino-4-chloromethylcoumarin (CMAC). CMAC fluorescence data were not used in the assay for detecting BT-20 cells. Only after the analysis using data from the specific and exclusion colors had been completed were the events identified as BT-20 cells checked for CMAC fluorescence. The putative BT-20 events were always found to be positive for CMAC fluorescence, which further increases confidence in the assay. Manual data analysis and an automated computer program were compared. Results were comparable with the manual and automated methods, but the automated "genetic algorithm" always found more

In a model study we seeded the pre-Bcell line REH into 250 million peripheral blood mononuclear cells (PBMC) at frequencies of and By flow cytometry we could detect the REH cells and found a background of about one event per 100 million PBMC. This sensitivity was achieved by removing four sources of false positive events, including nonspecific immunofluorescence, autofluorescence, background particles from previous experiments, and bursts of events during acquisition. To overcome limits to rare event detection imposed by nonspecific staining and autofluorescence, we used positive and negative selection for the REH cells. Another fluorochrome was added to stain the background cells and particles. In order to remove particles and background from previous experiments, a cleaning technique was developed and event bursts were removed from the analysis by developing an algorithm that screens the listmode data for events that were not Poisson distributed. o 1993 Wiley-Liss, Inc.