alamarBlue Assay: A Sensitive Fluorometric Method to Screen the Cytotoxic Effects of Artificial Tear Formulations on Metabolic Activity of Human Corneal Epithelial Cells

1. Human corneal epithelial cell culture

1. Grow immortalized HCEC in collagen-coated 75 cm² flasks with 20 mL of Dulbecco's modified Eagle medium/nutrient mixture F-12 (DMEM/F12) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C with 5% CO₂.
   NOTE: Shaking is not required.
2. Change the media every 2−3 days.

2. Preparation of cells for testing

1. After the cells are almost confluent remove the culture media.
2. Add 4−6 mL of cell disassociation solution to each 75 cm² flask. Incubate the cells at 37 °C until the cells detach (approximately 20−30 min), checking under the microscope periodically.
3. Add 2−6 mL of DMEM/F12 containing 10% FBS to each 75 cm² flask.
4. Transfer the contents of the flask to a 50 mL centrifuge tube.
5. Centrifuge the cells at 450−500 x g for 5 min. Pipet out the supernatant and resuspend the cells in pre-warmed media.
6. Count the cells using a hemocytometer. Calculate the volume of media that contains a total of 1 x 10⁵ cells.
7. Add 1 x 10⁵ cells to each well of a 48-well collagen-1 coated culture plate. Add enough media to the well so that the final volume of media in the well is 0.5 mL of culture DMEM/F12 with 10% FBS.
   NOTE: An artificial tear formulation may cause cytotoxicity that can be measured by a decrease in the metabolic activity of HCEC. The consistency of the data is dependent on adding the same number of HCEC to each well when seeding. The culture concentration that is recommended for 48 well plates is 1 x 10⁵. Because mammalian cells can settle in the centrifuge tube after resuspending, it is recommended to resuspend the cells by agitation frequently while seeding the plates with cells so that the cell suspension has the HCEC equally distributed.
8. Incubate the cells at 37 °C and 5% CO₂ for 24 h.

3. No desiccation protocol

1. Control procedure
   1. After the 24 h incubation remove the culture media from the plate.
   2. Immediately treat with 150 µL of a test formulation and media control solution.
   3. Incubate the cells at 37 °C and 5% CO₂ for 30 min.
   4. Remove the test solutions from the cells.
   5. Add 0.5 mL of 10% metabolic dye solution to test for metabolic activity. Incubate the cells for another 4 h at 37 °C and 5% CO₂.
   6. After the 4 h incubation period, remove 100 µL of metabolic dye solution from each well and place each 100 µL into a well of a 96-well plate.
   7. Measure the fluorescence of each well using a fluorescent plate reader (Table of Materials). Set the excitation wavelength at 540 nm and the emission wavelength at 590 nm.
2. No desiccation protocol (recovery procedure)
   1. Repeat steps 3.1.1−3.1.4.
   2. Add 0.5 mL of DMEM/F12 media to each well.
   3. Incubate one set of cultures for 18 h at 37 °C and 5% CO₂.
   4. Remove the media and add 0.5 mL of 10% metabolic dye solution. Repeat steps 3.1.6 and 3.1.7.