AlamarBlue bioassay for cellular investigation of UV-induced crystalline lens damage

Purpose: The use of the alamarBlue fluorescence dye for cellular study of UV-induced photodamage in cultured ocular lenses was examined by comparing the results from the fluorometric assay to lens optical quality using a scanning laser system to measure the focal lengths of the lenses following UVB treatment. Methods: Excised porcine lenses were cultured in M199 supplemented with 1% antibiotics and 4% porcine serum. After 1 week of pre-incubation at 37degreesC, baseline measurements were taken. Treated lenses were irradiated with a range of UVB radiant exposures from 0.019 to 0.076 J cm(-2). The lenses were maintained for a further 4 weeks, with measurements carried out every 48 h in the first 9 days post-UVB treatment and then once each week. At each measurement session, treated and control lenses were transferred into a 24-well plate, one lens per well containing the assay. The lenses were incubated for 50 min, after which fluorescence readings were taken with a plate reader. Results: Analyses showed significant (p < 0.05) inhibition of lens metabolic activity and optical function in the 0.038 and 0.076 J cm(-2) UVB treated lenses. Lenses treated with 0.019 J cm(-2) UVB did not exhibit any photodamage. Conclusions: These results suggest that the alamarBlue assay is useful for the in vitro study of UV-induced lens damage. The decrease in the capacity of treated lenses to reduce alamarBlue over time confirms that UVB photo-oxidation can cause diminution of viable lens epithelial and fibre cells. The results also suggest that the energy threshold for broadband UVB induced cataractogenesis in vitro ranges between 0.019 and 0.038 J cm(-2).