alamarBlue Assay: A Sensitive Fluorometric Method to Screen the Cytotoxic Effects of Artificial Tear Formulations on Metabolic Activity of Human Corneal Epithelial Cells
Published: April 30, 2023
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 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. Change the media every 2−3 days. 2. Preparation of cells for testing After the cells are almost confl…
Declarações
The authors have nothing to disclose.
Materials
| 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) |
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alamarBlue Assay: A Sensitive Fluorometric Method to Screen the Cytotoxic Effects of Artificial Tear Formulations on Metabolic Activity of Human Corneal Epithelial Cells. J. Vis. Exp. (Pending Publication), e21238, doi: (2023).