Korean J Ophthalmol > Volume 10(2); 1996 > Article
Korean Journal of Ophthalmology 1996;10(2):63-67.
DOI: https://doi.org/10.3341/kjo.1996.10.2.63    Published online December 30, 1996.
The effect of dichlorotriazinyl aminofluorescein on human keratocytes in vitro.
Won Ryang Wee, Roya Rofougaran, Alireza Pakkar, Seiji Hayashi, Peter J McDonnell
1Department of Ophthalmolog, Seoul National University COllege of Medicine, Seoul, Korea.
2Doheny Eye Institute and the Department of Ophthalmology, University of Southern California School of Medicine, Los Angeles, California, USA.
Dichlorotriazinyl aminofluorescein (DTAF) has been used to stain corneal stromal collagen as part of in vivo animal experiments for many years. Toxicity of this drug, if present, might alter the observed wound healing. To determine if this drug has any deleterious effect on keratocytes, we evaluated it in vitro. Human keratocytes prepared in 24-well plates were exposed to varying concentrations of DTAF (10(-4), 10(-3), 10(-2), 1, 10, 10(2) microgram/ml). Exposure times of 1 hour and 24 hours at each concentration of DTAF were evaluated. The cell number was measured 1 and 3 days after initiation of exposure to DTAF using a Coulter counter. Keratocyte proliferation was not affected by 1-hour exposure to DTAF, but keratocyte proliferation measured 3 days after initiation of exposure to DTAF for 24 hours was inhibited in a dose-dependent manner (p = 0.02) and was significantly inhibited at concentrations of 10 and 100 microgram/ml (p < 0.05). Fluorescent microscopy showed binding of DTAF to keratocytes. We have demonstrated that prolonged exposure to DTAF inhibits proliferation of cultured keratocytes. These results suggest that DTAF-induced cytotoxicity may alter net production of collagen in the corneal stroma in animal models.
Key Words: dichlorotriazinyl aminofluorescein;collagen;keratocyte;cytotoxicity

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