Imaging flow cytometry methods for quantitative analysis of label-free crystalline silica particle interactions with immune cells

Bradley Vis; Jonathan J. Powell; Rachel E. Hewitt; Bradley Vis; Jonathan J. Powell; Rachel E. Hewitt

doi:10.3934/biophy.2020012

AIMS Biophysics

2020, Volume 7, Issue 3: 144-166. doi: 10.3934/biophy.2020012

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Imaging flow cytometry methods for quantitative analysis of label-free crystalline silica particle interactions with immune cells

Biomineral Research Group, Department of Veterinary Medicine, University of Cambridge, Madingley Rd, Cambridge CB3 0ES, UK

Received: 27 February 2020 Accepted: 21 May 2020 Published: 26 May 2020

Exposure to respirable fractions of crystalline silica quartz dust particles is associated with silicosis, cancer and the development of autoimmune conditions. Early cellular interactions are not well understood, partly due to a lack of suitable technological methods. Improved techniques are needed to better quantify and study high-level respirable crystalline silica exposure in human populations. Techniques that can be applied to complex biological matrices are pivotal to understanding particle-cell interactions and the impact of particles within real, biologically complex environments. In this study, we investigated whether imaging flow cytometry could be used to assess the interactions between cells and crystalline silica when present within complex biological matrices. Using the respirable-size fine quartz crystalline silica dust Min-u-sil® 5, we first validated previous reports that, whilst associating with cells, crystalline silica particles can be detected solely through their differential light scattering profile using conventional flow cytometry. This same property reliably identified crystalline silica in association with primary monocytic cells in vitro using an imaging flow cytometry assay, where darkfield intensity measurements were able to detect crystalline silica concentrations as low as 2.5 µg/mL. Finally, we ultilised fresh whole blood as an exemplary complex biological matrix to test the technique. Even after the increased sample processing required to analyse cells within whole blood, imaging flow cytometry was capable of detecting and assessing silica-association to cells. As expected, in fresh whole blood exposed to crystalline silica, neutrophils and cells of the monocyte/macrophage lineage phagocytosed the particles. In addition to the use of this technique in in vitro exposure models, this method has the potential to be applied directly to ex vivo diagnostic studies and research models, where the identification of crystalline silica association with cells in complex biological matrices such as bronchial lavage fluids, alongside additional functional and phenotypic cellular readouts, is required.
- crystalline silica particles,
- quartz dust,
- imaging flow cytometry,
- macrophages,
- neutrophils,
- phagocytes,
- label-free
Citation: Bradley Vis, Jonathan J. Powell, Rachel E. Hewitt. Imaging flow cytometry methods for quantitative analysis of label-free crystalline silica particle interactions with immune cells[J]. AIMS Biophysics, 2020, 7(3): 144-166. doi: 10.3934/biophy.2020012

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Abstract

Exposure to respirable fractions of crystalline silica quartz dust particles is associated with silicosis, cancer and the development of autoimmune conditions. Early cellular interactions are not well understood, partly due to a lack of suitable technological methods. Improved techniques are needed to better quantify and study high-level respirable crystalline silica exposure in human populations. Techniques that can be applied to complex biological matrices are pivotal to understanding particle-cell interactions and the impact of particles within real, biologically complex environments. In this study, we investigated whether imaging flow cytometry could be used to assess the interactions between cells and crystalline silica when present within complex biological matrices. Using the respirable-size fine quartz crystalline silica dust Min-u-sil® 5, we first validated previous reports that, whilst associating with cells, crystalline silica particles can be detected solely through their differential light scattering profile using conventional flow cytometry. This same property reliably identified crystalline silica in association with primary monocytic cells in vitro using an imaging flow cytometry assay, where darkfield intensity measurements were able to detect crystalline silica concentrations as low as 2.5 µg/mL. Finally, we ultilised fresh whole blood as an exemplary complex biological matrix to test the technique. Even after the increased sample processing required to analyse cells within whole blood, imaging flow cytometry was capable of detecting and assessing silica-association to cells. As expected, in fresh whole blood exposed to crystalline silica, neutrophils and cells of the monocyte/macrophage lineage phagocytosed the particles. In addition to the use of this technique in in vitro exposure models, this method has the potential to be applied directly to ex vivo diagnostic studies and research models, where the identification of crystalline silica association with cells in complex biological matrices such as bronchial lavage fluids, alongside additional functional and phenotypic cellular readouts, is required.

Abbreviation PBMC: peripheral blood mononuclear cells; ISX: Imagestream X; DF: darkfield; BF: brightfield; BDI: bright detail intensity; SSC: side scatter; FS: forward scatter; AUL: area upper limit; ALL: area lower limit; gradient RMS: gradient root mean square; WB: whole blood; WEL: workplace exposure limit; NET: neutrophil extracellular trap; 7-AAD: 7-Aminoactinomycin D; PM, PM and PM: Particulate matter of less than 0.1, 2.5 or 10 µm median aerodynamic diameters;
Acknowledgments

This study was funded by UK Medical Research Council, Grant number MR/R005699/1. The authors wish to thank the Volunteer Studies and Clinical Services Team at MRC EWL for recruitment and written consent of volunteers and the volunteers' blood collection.

Conflict of interest

The authors declare no conflicts of interest.

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