New Laboratory Protocol to Determine the Oxidative Stress Profile of Human Nasal Epithelial Cells Using Flow Cytometry

Abstract

Several studies have shown the importance of oxidative stress (OS) in respiratory disease pathogenesis. It has been reported that the nasal epithelium may act as a surrogate for the bronchial epithelium in several respiratory diseases involving OS. However, the sample yields obtained from nasal biopsies are modest, limiting the number of parameters that can be determined. Flow cytometry has been widely used to evaluate cellular OS profiles. It has the advantage that analyses can be performed using a small amount of sample. Therefore, we aimed to set up a new method based on flow cytometry to assess the oxidative profile of human nasal epithelial cells which could be used in research on respiratory diseases. Levels of total nitric oxide, superoxide anion, peroxynitrite, and intracellular peroxides were measured. Reduced thiol levels, such as antioxidant-reduced glutathione and oxidative damaged lipids and proteins, were also analysed. The intracellular calcium levels, plasma membrane potential, apoptosis, and percentage of live cells were also studied. Finally, a strategy to evaluate the mitochondrial function, including mitochondrial hydrogen peroxide, superoxide anion, mitochondrial mass, and membrane potential, was set up. Using small amounts of sample and a non-invasive sampling technique, the described method enables the measurement of a comprehensive set of OS parameters in nasal epithelial cells, which could be useful in research on respiratory diseases.

Keywords: flow cytometry; nasal epithelium; oxidative stress; rare respiratory diseases; reactive oxygen species.

Figure 1

Gating strategy used to select the population of interest using a FACS Verse cytometer. (a) Nasal epithelial cells were selected by side scatter characteristic (SSC) and forward scatter characteristic (FSC) density plots. (b) A gate was applied to identify specific populations of individual cells. Each dot represents one nasal epithelial cell that passed through the cytometer laser. (c) Dead cells were identified and excluded from further analysis using either DAPI or PI.

Figure 2

Kinetic measurements of nitric oxide (NO) assays using flow cytometry. (a) Dot blot acquired from kinetic measurements. Live nasal epithelial cells were gated according to FSC and SSC, and the gated events were plotted against a FITC-A channel (in this case, DAF-FM DA) and time. (b) Table showing gating percentages and fluorescence levels increasing over time.

Figure 3

Flow cytometric analysis of peroxynitrite generation kinetics in nasal epithelial cells. (a) Dot blot acquired from kinetic measurements. Live nasal epithelial cells were gated according to FSC and SSC, and the gated events were plotted against a FITC-A channel (in this case DHR123) and time. Acquisition was paused at two points to add plumbagin (PB) (an O₂⁻ provider) and NOR-1 (a NO inductor). (b) Table showing gating percentages and fluorescence levels increasing over time.

Figure 4

Flow cytometric analysis of intracellular calcium (iCa2+) generation kinetics in nasal epithelial cells. Intracellular calcium was measured using FLUO-4. Human nasal cells were gated accordingly to SSC and FSC. Dead cells were excluded using DAPI. Gating percentages and iCa2+ levels are shown.

Figure 5

Evaluation of lipid peroxidation using BODIPY 665/676 C11. (a) Nasal epithelial cells were selected based on morphology and PI staining to exclude dead cells. (b) and (c) show the ratios of oxidised vs. reduced lipids.

Figure 6

Apoptosis and cell death determination using Annexin V and propidium iodide (PI) staining. Nasal epithelial cells were selected based on morphology. Apoptosis status was determined by Annexin V staining. Cell death was determined by PI staining. Gating methods for Annexin V−/PI− (live), Annexin V+/PI− (early apoptosis), Annexin V−/PI+ (necrosis), and Annexin V+/PI+ (late apoptosis) are shown.