A Simple Fluorescent Cell-Type-Specific Labeling Method for Cocultured Cell Flow Cytometry Analysis

Introduction: Coculture models have been extensively used for assessing the toxicity of fibers 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 might not be compatible with assays requiring living cells such as a particle-induced oxidative stress assay by flow cytometry. Materials and Methods: Human alveolar epithelial cells (A549) were specifically labeled with cell proliferation dye—eFluor™ 670—before being mixed with phorbol ester-differentiated Tohoku Hospital Pediatrics-1 cells (used as macrophages). The coculture 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). Results: A549 cell treatment with a noncytotoxic concentration of an eFluor 670 probe allowed labeled and unlabeled 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. Conclusions: This study showed that specific labeling of live cells before coculture setup allows assays such as oxidative stress assessment by flow cytometry to be conducted on different live cell types from the same coculture models without the use of cell-type-specific markers. Such assays would be of great value in any kind of multiple cell type model in which the effects of chemicals on a given cell population is sought.