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

Abstract

Source: Rangarajan, R. et al. Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation. J. Vis. Exp. (2020)

This video describes an in vitro fluorometric assay to assess the effects of artificial tear formulations on the cellular metabolic activity of corneal epithelial cells using resazurin-based Alamar blue solution. A non-toxic formulation maintains the cells in a healthy, metabolically active state, facilitating the reduction of resazurin into a highly fluorescent resorufin.

Protocol

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 unti....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
96 well plate	costar	3370
alamarBlue	Fisher Scientific	dal 1025
Corning 48 Well plates	Corning	354505	This is the BioCoat brand collagen coated
DMEM/F12 with L-glutamine and 15 mM HEPES	Gibco	11039-021	There is No Phenol Red in this media
Fetal Bovine Serum	Hycone	SH30396.03
Human Corneal Epithelial Cells	University of Ottawa	N/A	SV40-immortalized human corneal epithelial cells from Dr. M. Griffith (Ottawa Eye Research Institute, Ottawa, Canada) and have been characterized Griffith M, Osborne R, Munger R, Xiong X, Doillon CJ, Laycock NL, Hakim M, Song Y, Watsky MA. Functional human corneal equivalents constructed from cell lines. Science. 1999;286:2169–72.
TrypLE Express (cell disassociation solution)	Fisher Scientific	12605036
Cytation 5	BioTek	CYT5MPV	Can read fluorescence from 280 - 700 nm (for assay 540/590)