To test the ability of LED arrays to identify photosensitizing small molecules capable of cellular toxicity, we chose compounds with known optical properties: tetra-methyl-rhodamine methyl ester perchlorate (TMR), tetra-methyl-rosamine chloride (TMRs), and malachite green carbinol HCl (MG) (T5428-25 mg, T-1823, and Aldrich 213020-25G, respectively; all from Sigma-Aldrich, St. Louis, MO). TMR accumulates in mitochondria and, on photoactivation, induces ROS formation that causes loss of mitochondrial membrane potential and eventual phototoxic apoptosis.
22 TMRs is a triphenylmethane dye structurally similar to TMR and exerts cellular phototoxicity by the same mechanism.
2,23 MG is not toxic to cells at a light dose of 40 J/cm
2 making it a good negative control.
24 All compounds were dissolved in DMSO and stored at −20°C, protected from light. Spectral data on these compounds were obtained from Invitrogen-Molecular Probes (Carlsbad, CA), Web site (
http://www.invitrogen.com/site/us/en/home/support/research-tools/fluorescence-spectraviewer.reg.us.html). Chemical structures were drawn in computer software (MDL ISIS/Draw, ver. 2.5; Symyx Corp., Sunnyvale, CA). TMR and TMRs, which are activated efficiently by green light at 526 nm, were compared to MG, which absorbs in the red but not significantly in the green portion of the visible spectrum. HEK293S cells
25 (∼50,000 cells/well) were plated onto 96-well, black-walled, cell culture–treated plates (Optilux; BD-Falcon) and then incubated overnight at 37°C in a 5% CO
2/95% air mixture in a cell culture incubator (Forma Scientific, Marietta, OH). Cell culture medium was DMEM/F12 with 10% heat-inactivated calf serum plus antibiotics (Invitrogen). Test compounds were added to a final test concentration of 1 mM in the culture medium. The plate was then exposed to the stipulated LED arrays for 0, 1, 2, or 4 hours. The following day, SYTOX Green (S7020; Invitrogen-Molecular Probes) was added to the cell culture medium to a final concentration of 5 μM. A quantitative imaging platform was used in this cytotoxicity assay. This platform has been described in part (Butler MC, et al.
IOVS 2007;48:ARVO E-Abstract 4608; Butler MC, et al.
IOVS 2008;49:ARVO E-Abstract 5342). The plates were imaged with an epifluorescence microscope (TE-300; Nikon, Tokyo, Japan) equipped with a mercury halogen lamp, 20× (NA 0.4) plan fluor objective lens, FITC cube, and monochrome cooled 12-bit digital CCD camera (Evolution Qe
i; Media Cybernetics, Silver Spring, MD). The SYTOX Green images were obtained with a 20-ms exposure. The images were recorded, statistically segmented, and quantified by using identical parameters throughout. The data were plotted with total fluorescence emission (523 nm) from the SYTOX Green versus time-exposed to the LED array for each of the various compounds and control agents. Data were analyzed (IP-Laboratories software, ver. 3.7; Scanalytics, Inc, Rockville, MD) and the data were plotted (Origin 6.1 software; OriginLab Corp., Northampton, MA).