title: Phagocytosis-coupled flow cytometry for detection and size discrimination of anionic polystyrene particles.
abstract: Flow cytometry was evaluated for its capacity to detect and distinguish a wide size range (20-2000 nm) of fluorescent polystyrene particles (PSPs). Side scatter and fluorescence parameters could predict dispersed PSP sizes down to 200 nm, but the forward scatter parameter was not discriminatory. Confocal microscopy of flow-sorted fractions confirmed that dispersed PSPs appeared as a single sharp peak on fluorescence histograms, whereas agglomerated PSPs were detected as smaller adjacent peaks. Particles as small as 200 nm could also be detected by flow cytometry after they were first phagocytized by J774A.1 murine macrophages. Confocal microscopy demonstrated that these PSPs were internalized within the cytoplasm. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and calcein-AM (acetoxymethyl ester) assays showed that they were not cytotoxic. Internalized PSP size correlated to both cellular side scatter (R(2)=0.9821) and fluorescence intensity (R(2)=0.9993). Furthermore, PSPs of various sizes could be distinguished when J774A.1 cells were loaded with a single size of PSP and mixed with cells containing other sizes. However, spectra of cells loaded with a mixture of PSP sizes resembled those containing only the largest PSP. These data demonstrate the capacity and limitations of phagocytosis-coupled flow cytometry to distinguish between dispersed and agglomerated states and detect a wide size range of particles.
authors.affiliation: Biotechnology Laboratory, Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Environmental Health Centre, Health Canada, Ottawa, Ontario K1A 0K9, Canada.
authors.forename: Emily
authors.initials: E
authors.lastname: Mutzke
authors.affiliation: Flow Cytometry Laboratory, Bureau of Chemical Safety, Health Products and Food Branch, Frederick G. Banting Building, Health Canada, Ottawa, Ontario K1A 0K9, Canada.
authors.forename: Emily
authors.initials: E
authors.lastname: Chomyshyn
authors.affiliation: Biotechnology Laboratory, Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Environmental Health Centre, Health Canada, Ottawa, Ontario K1A 0K9, Canada.
authors.forename: Kathy C
authors.initials: KC
authors.lastname: Nguyen
authors.affiliation: Biotechnology Laboratory, Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Environmental Health Centre, Health Canada, Ottawa, Ontario K1A 0K9, Canada.
authors.forename: Maria
authors.initials: M
authors.lastname: Blahoianu
authors.affiliation: Biotechnology Laboratory, Environmental Health Science and Research Bureau, Healthy Environments and Consumer Safety Branch, Environmental Health Centre, Health Canada, Ottawa, Ontario K1A 0K9, Canada. Electronic address: azam.tayabali@hc-sc.gc.ca.
authors.forename: Azam F
authors.initials: AF
authors.lastname: Tayabali
mesh_terms: D000818:Animals; D000838:Anions / Q000737:chemistry*; D016923:Cell Death; D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D042541:Intracellular Space / Q000378:metabolism; D051379:Mice; D008853:Microscopy; D010316:Particle Size; D010587:Phagocytosis; D011137:Polystyrenes / Q000737:chemistry*
chemical_list: D000838:Anions; D011137:Polystyrenes
keywords: Agglomeration; Fluorescence; Macrophage; Microparticles; Nanoparticles; Phagocytosis