Guidelines for the use of flow cytometry and cell sorting in immunological studies^*

Abstract

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

Rigor and Reproducibility of Cytometry Practices for Immuno‐Oncology: A multifaceted challenge

“…Data from studies involving multi-centers, each performing its own supervised analysis, have indicated that manual gating introduces the largest variable in standardized experiments (17)(18)(19)(20)(21)40). Data from studies involving multi-centers, each performing its own supervised analysis, have indicated that manual gating introduces the largest variable in standardized experiments (17)(18)(19)(20)(21)40).…”

“…Most researchers will begin with the manufacturer recommended titration and test multiple doses until an optimal stain index is achieved (20)(21)(22)(23)(24). Initial titration of antibodies is a wellestablished criterion for good experimental design.…”

An integrated enrichment system to facilitate isolation and molecular characterization of single cancer cells from whole blood

“…Others have shown this approach to be useful in clinical studies isolating and characterizing rare cells (27)(28)(29). The removal of debris and depletion of WBCs effectively reduced the event rate to the sorter, and this is a feature that has been reported to enhance the sort efficiency of rare cancer cell events (9,33). The AF chip is able to recover cells at or larger than WBCs with high recovery (Supporting Information Fig.…”

“…As peripheral blood CTCs are present at very low frequency (1-100 cells per ml of whole blood), it is useful to enrich them prior to FACS analysis and sorting not only to avoid coincidence events (8) but also to insure optimal sort efficiency (9). As peripheral blood CTCs are present at very low frequency (1-100 cells per ml of whole blood), it is useful to enrich them prior to FACS analysis and sorting not only to avoid coincidence events (8) but also to insure optimal sort efficiency (9).…”

Relevance of Antibody Validation for Flow Cytometry

“…In the course of sample preparation for flow cytometry, the cells must be fixed and permeabilized to make the target epitopes available for antibody binding. and VII.15 in the study by Cossariza et al (59). A particular sample preparation (fixation and permeabilization) protocol is the key to successful staining for intracellular cytometry.…”