title: Detection of fluorescent nanoparticle interactions with primary immune cell subpopulations by flow cytometry.
abstract: Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.
authors.affiliation: Center for Micro-BioRobotics @SSSA, Istituto Italiano di Tecnologia.
authors.forename: Olimpia
authors.initials: O
authors.lastname: Gamucci
authors.affiliation: Center for Micro-BioRobotics @SSSA, Istituto Italiano di Tecnologia; Department of Biology, University of Pisa.
authors.forename: Alice
authors.initials: A
authors.lastname: Bertero
authors.affiliation: Center for Biomolecular Nanotechnologies @UniLe, Istituto Italiano di Tecnologia.
authors.forename: Maria Ada
authors.initials: MA
authors.lastname: Malvindi
authors.affiliation: Center for Biomolecular Nanotechnologies @UniLe, Istituto Italiano di Tecnologia.
authors.forename: Stefania
authors.initials: S
authors.lastname: Sabella
authors.affiliation: Center for Biomolecular Nanotechnologies @UniLe, Istituto Italiano di Tecnologia.
authors.forename: Pier Paolo
authors.initials: PP
authors.lastname: Pompa
authors.affiliation: Center for Micro-BioRobotics @SSSA, Istituto Italiano di Tecnologia.
authors.forename: Barbara
authors.initials: B
authors.lastname: Mazzolai
authors.affiliation: Center for Micro-BioRobotics @SSSA, Istituto Italiano di Tecnologia; Giuseppe.Bardi@iit.it.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Bardi
mesh_terms: D000328:Adult; D000818:Animals; D001672:Biocompatible Materials / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000737:chemistry / Q000166:cytology*; D051379:Mice; D008822:Mice, Transgenic; D017628:Microglia / Q000737:chemistry / Q000166:cytology*; D008875:Middle Aged; D053758:Nanoparticles / Q000737:chemistry* / Q000378:metabolism; D012822:Silicon Dioxide / Q000097:blood / Q000737:chemistry; D055815:Young Adult
chemical_list: D001672:Biocompatible Materials; D005456:Fluorescent Dyes; D012822:Silicon Dioxide; D016650:Fluorescein-5-isothiocyanate
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