Korean J Ophthalmol.  1996 Dec;10(2):63-67. 10.3341/kjo.1996.10.2.63.

The effect of dichlorotriazinyl aminofluorescein on human keratocytes in vitro

• Wee WR
• Rofougaran R
• Pakkar A
• Hayashi S
• McDonnell

• KMID: 1735160
• DOI: http://doi.org/10.3341/kjo.1996.10.2.63

Abstract

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.

Keyword

dichlorotriazinyl aminofluorescein; collagen; keratocyte; cytotoxicity

MeSH Terms

Cell Count
Cell Division/drug effects
Cells, Cultured
Corneal Stroma/cytology/*drug effects
Fibroblasts/cytology/drug effects
Fluoresceins/*pharmacology/toxicity
Fluorescent Dyes/*pharmacology/toxicity
Humans
Microscopy, Fluorescence
Fluoresceins
Fluorescent Dyes