Phenotypic and functional characterization of in vitro cultured human mast cells

Abstract

This study discloses that culturing HuMC from CD34 progenitors yields 2 phenotypically distinct cell populations that display a greatly similar response upon cross-linking of IgE/FcεRI complexes. © 2016 International Clinical Cytometry Society.

Influence of IL‐6, IL‐33, and TNF‐α on human mast cell activation: Lessons from single cell analysis by flow cytometry

“…Therefore, we took advantage of our flow cytometric technique that allows to combine immunophenotyping and quantification of degranulation by individual cells to study the effect of these proinflammatory cytokines on IgE-and MRGPRX2-dependent activation of cultured MCs that, as already reported in the Journal, express a connective tissue phenotype (MC TC ) (26). Besides, our current knowledge is mainly based on data gathered by mediator release tests from which the results merely represent the average of all stimulated cells.…”

“…Immunophenotyping Cultured human MCs were immunophenotyped as described elsewhere (26). Briefly, human MCs were stained for surface makers with monoclonal antihuman CD45-peridinin chlorophyll (CD45-PerCP) (BioLegend, San Diego, USA), antihuman CD117-phycoerythrin (CD117-PE) (BD Biosciences), or antihuman CD117allophycocyanin (CD117-APC) (BD Biosciences) when in combination with antihuman FcERI-phycoerythrin (FcERI-PE) (eBioscience, San Diego, USA), antihuman CD203c-allophycocyanin (CD203c-APC) (Pharmingen, BD Biosciences, Erembodegem, Belgium), and antihuman CD63-fluorescein isothiocyanate (CD63-FITC) (BD Biosciences).…”

“…Actually, it has been shown that IL-6 and IL-33 promote IgE-dependent degranulation as measured by release of cytokines and/or mediators in the supernatant (13)(14)(15). Recently, we developed a flow cytometric assay that enables to study degranulation patterns of individual basophils (7,24,25) and individual cultured human MCs (26). In contrast, data about the effects of these cytokines on human MCs are scarce and have mainly been gathered using mediator release assays in the supernatant of cultured human MCs (17)(18)(19)(20)(21)(22)(23).…”

“…Besides, IL-33 has been shown to increase cytokine and chemokine production after IgE-independent stimulation via IgG immune complexes (16). For this purpose, anaphylactic degranulation of MCs is analyzed by flow cytometry (24)(25)(26)(27). Results from these assays only reflect the average of all stimulated cells and do not allow to obtain data from individual cells.…”

“…Results from these assays only reflect the average of all stimulated cells and do not allow to obtain data from individual cells. Recently, we developed a flow cytometric assay that enables to study degranulation patterns of individual basophils (7,24,25) and individual cultured human MCs (26). The most important goal of this assay is that it allows to simultaneously analyze the immunophenotype and histamine release.…”