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.

Noxic effects of polystyrene microparticles on murine macrophages and epithelial cells

“…The ingestion of particles has in principle been reported for sizes up to 10 µm for J774A.1, but particle surface modifications with e.g. IgG or carboxyl groups were necessary in such cases, and ingestion rates were still rather low 43 , 68 , 70 – 72 . For the liver macrophage cell line ImKC, a similar trend as for J774A.1 was observed.…”

Critical issues in genotoxicity assessment of TiO2 nanoparticles by human peripheral blood mononuclear cells

“…Because PBMCs are composed by both monocytes and lymphocytes, we evaluated by flow cytometry the ability of the different cell subpopulations to interact with TiO 2 particles. This approach, based on SSC signal intensity, has been applied successfully in several studies indicating its suitability for this purpose (Mutzke, Chomyshyn, Nguyen, Blahoianu, & Tayabali, ; Suzuki, Toyooka, & Ibuki, ; Zucker et al, ). Data clearly indicated that TiO 2 particles interacted mainly with the monocyte cell subpopulation, with negligible interaction with the lymphocyte subpopulation (Herd et al, ; Thurn et al, ).…”

Differential Phagocytic Properties of CD45low Microglia and CD45high Brain Mononuclear Phagocytes—Activation and Age-Related Effects

“…As compared to peripheral tissue macrophages, microglia have lower phagocytic capacity which can be augmented following activation by pro-inflammatory stimuli ( 14 , 15 ). Measurement of phagocytic capacity of microglia can be performed by in vitro engulfment assays using fluorescent probe-labeled particles, Aβ aggregates, opsonized bacteria, or latex particles followed by immunofluorescence microscopy ( 14 , 16 , 17 ). These assays can introduce sampling biases by exclusion of non-adherent cells in a cell type-like BV2 that has significant proportions of live floating cells.…”