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Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability.

ABSTRACT: PURPOSE:To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability. METHODS:B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated by flow cytometry in order to derive software filters based on morphological parameters of separate cell populations with MFI and FlowCAM. The filter sets were used to assess cell viability in the measured samples. RESULTS:All techniques gave fairly similar cell concentration values over the whole incubation period. MFI showed to be superior with respect to precision, whereas FlowCAM provided particle images with a higher resolution. Moreover, both FIM methods were able to provide similar results for cell viability as the conventional methods (hemocytometry and automated cell counting). CONCLUSION:FIM-based methods may be advantageous over conventional cell methods for determining total cell concentration and cell viability, as FIM measures much larger sample volumes, does not require labeling, is less laborious and provides images of individual cells.

SUBMITTER: Sediq AS

PROVIDER: S-EPMC5976703 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability.

Sediq A S AS Klem R R Nejadnik M R MR Meij P P Jiskoot Wim W

Pharmaceutical research 20180530 8

<h4>Purpose</h4>To investigate the potential of two flow imaging microscopy (FIM) techniques (Micro-Flow Imaging (MFI) and FlowCAM) to determine total cell concentration and cell viability.<h4>Methods</h4>B-lineage acute lymphoblastic leukemia (B-ALL) cells of 2 different donors were exposed to ambient conditions. Samples were taken at different days and measured with MFI, FlowCAM, hemocytometry and automated cell counting. Dead and live cells from a fresh B-ALL cell suspension were fractionated ...[more]

PMID: 29846807

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Project description: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 ?g/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.

Dataset Information

Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability.

Publications

Label-Free, Flow-Imaging Methods for Determination of Cell Concentration and Viability.

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