Figure 6.

Investigation of the ability to detect the presence of reactive oxygen species (ROS) using the fluorescent probe 2′,7′-dichlorfluorecein-diacetate (DCFH₂-DA) (i.e. the DCFH-DA assay) in the 3D in vitro triple cell co-culture of the human lung epithelial tissue barrier following detachment from micro-porous membrane insert via EDTA-Trypsin treatment. The ROS response was measured for (A) the total cell suspension via one-colour flow cytometry (FACS), or (B) for each specific cell type of the co-culture system via four-colour FACS. In both (A and B), data from one- and four-colour FACS shows the overall ROS response in the co-culture suspension following 4 hours exposure at 37 °C, 5% CO₂ to either complete medium (negative control) or tert-Butyl Hydrogen Peroxide (tBHP) at [0.04 mg/mL]. Following the exposure period, the co-culture cell suspension was treated with 1:10 dilution of 1 mM DCFH-DA (488 nm). A DCFH-DA treated control (DCFH-ve) was also performed to denote any non-specific fluorescence within the sample. After implementing the specific gating strategy for the co-culture suspension (SI Fig. 1b) (C) indicates the gating strategy used to understand a negative and positive response from the multi-cell suspension using the DCFH-DA assay. All data was analysed using FlowJo (Version 10, TreeStar, USA). Data expressed is the mean ± standard error of the mean (SEM) of the %Frequency. In (A and B) all data expressed is the mean ± standard error of the mean (SEM). Experimentation was repeated on three separate occasions in triplicate (n = 3).