This study investigates the effects of artificial tear formulations on corneal epithelial cells (CECs) using the alamarBlue assay. It highlights the importance of maintaining a healthy tear film to prevent cellular damage and infection.
Tear film - the protective fluid covering the eye - helps lubricate and maintain ocular corneal epithelial cells, CECs, in a healthy, metabolically active state. A compromised tear film, as in dry eye disease, can cause tear evaporation from CECs, increasing their susceptibility to infection and inflammatory damage.
Artificial tear formulations stabilize the tear film and improve water retention, preventing cell dehydration.
To screen the cytotoxic effects of a test artificial tear formulation on CECs using alamarBlue assay, begin with an adherent culture of human-derived CECs in suitable media. Discard the media.
Incubate the cell culture with a specific concentration of the artificial tear formulation. The tear formulation forms a coating over the cells. Depending on the cumulative effect of the constituting ingredients, cells may experience a loss in metabolic activity.
Next, add alamarBlue solution containing resazurin, a blue-colored, weakly fluorescent, non-toxic, water soluble dye.
Resazurin diffuses into the cells and gets irreversibly reduced in metabolically active cells primarily by mitochondrial reductases as it accepts electrons from the electron donors of the respiration-metabolic processes, forming a pink-colored, highly fluorescent resorufin, which freely moves out of the cells into the solution. Transfer the resorufin-containing solution to a fresh multi-well plate.
Using a fluorescence plate reader, measure the fluorescence intensity. A low fluorescence intensity value indicates a low cellular metabolic activity, suggesting undesirable effects of the tear formulation on CECs.
Remove the culture media from the wells, and immediately treat the cells with 150 microliters of a test formulation or media control solution. Then, incubate the cells for 30 minutes.
Remove the test solution from the cells, and add 0.5 milliliters of 10% metabolic dye solution. Incubate the cells for another four hours.
After the incubation, remove 100 microliters of dye solution from each well and transfer it to a 96-well plate. Use a plate reader to measure the fluorescence of each well, setting the excitation to 540 nanometers and emission to 590 nanometers.
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