Phagocytosis-coupled flow cytometry for detection and size discrimination of anionic polystyrene particles
- DOI
- Language of the publication
- English
- Date
- 2015-05-05
- Type
- Article
- Author(s)
- Mutzke, Emily
- Chomyshyn, Emily
- Nguyen, Kathy C.
- Blahoianu, Maria
- Tayabali, Azam F.
- Publisher
- Elsevier
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 (R2 = 0.9821) and fluorescence intensity (R2 = 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.
Plain language summary
Nanometer and micrometer sized particles could pose a risk to human health. However, in order to interpret the toxicity of these particles, it is necessary to understand some basic physical characteristics of these particles. Towards this goal, Health Canada is developing methods to efficiently characterise particles of various compositions. This paper describes the use of an existing cell counting instrument called a flow cytometer that was used to provide size and clumping information of large numbers of fluorescent nanometer and micrometer sized particles. Separate fractions were obtained that contained either individual or clumped particles. The detection sensitivity of this method was enhanced by first allowing immune cells to internalise the particles. Particles as small as 20 nm were detected within cells by monitoring either the fluorescent characteristics of the particle, or changes in the granularity of the immune cell. Finally, immune cells that had taken up one size of particle were distinguished from those containing another size of particle, even after the cells were mixed together. This study demonstrated that flow cytometry was a useful tool for providing physical information about particles as they exist in suspension, as well as within immune cells. This data ultimately provided essential information about how particles behave outside and inside mammalian cells. Future work will examine what other types of materials can be detected using this method.
Subject
- Health,
- Health and safety