A simple fluorescent cell-type specific labelling method for co-cultured cell flow cytometry analysis

Introduction:<br/>
Co-culture models have been extensively used for assessing the toxicity of fibres and particles. However, once cell lines are mixed in the same tissue culture well, it is difficult to evaluate differential cell toxicity without using specific cell markers, which are not compatible with assays requiring living cells such as a particle-induced oxidative stress assay by flow cytometry.<br/>
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Materials and Methods:<br/>
Human alveolar epithelial cells (A549) were specifically labelled with cell proliferation dye - eFluor 670 - prior to being mixed with phorbol ester-differentiated THP-1 (used as macrophages). The co-culture model allowed the toxicity of crystalline silica DQ-12 and DQ-12-PVNO (particles were coated with polyvinylpyridine-N-oxide, a molecule used to quench particle surface reactivity) to be assessed. Particle-induced oxidative stress was evaluated by flow cytometry using a 2',7'-dichlorodihydrofluorescein diacetate probe (H2DCFDA).<br/>
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Results: <br/>
A549 cell treatment with a non-cytotoxic concentration of an eFluor 670 probe allowed labelled and unlabelled cells to be differentiated using flow cytometry. Cellular oxidative stress induced by phorbol ester or DQ-12 detected by H2DCFDA was not affected by eFluor 670 probe cell treatment.<br/>
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Conclusions:<br/>
This work showed that specific labelling of live cells prior to co-culture setup allows assays such as oxidative stress assessment by flow cytometry to be conducted on different live cell types from the same co-culture models without the use of cell-type-specific markers. Such assays would be of great value in any type of multiple cell type model in which the effects of chemicals on a given cell population is sought.