Effect of Photofrin on DNA Strand Breaks and Base Oxidation in HaCaT Keratinocytes: A Comet Assay Study6, The

Photochemistry and Photobiology, Jan 2004 by Woods, J A, Traynor, N J, Brancaleon, L, Moseley, H

MATERIALS AND METHODS

Materials. All chemicals and cell culture reagents were obtained from the Sigma Aldrich Chemical Co. (Poole, UK) unless otherwise stated. Cell culture plastics were supplied by Costar (Cambridge, UK). Photofrin was obtained from Axcan Pharma Inc. (Mont-Saint-Hilaire, QC, Canada). Endonuclease III and FPG protein were kindly supplied by Professor Andrew Collins (Rowett Research Institute, Aberdeen, Scotland, UK).

Cell maintenance. Human HaCaT keratinocytes (kindly provided to our laboratory by Professor N. Fusenig, Heidelberg) were maintained in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum and 2 mM L-glutamine. Cells were grown in a humidified incubator in an atmosphere of 5% CO2 and 95% air at 37�C and passaged every 7-10 days. The cells were grown in the absence of antibiotics and were screened for mycoplasme using a Hoechst staining method. For all experiments, cells were seeded at a density of 1 � 10^sup 5^ cells/cm^sup 2^ in tissue culture plates. Liquid volumes were scaled accordingly.

Preparation of compounds. Photofrin slock solutions were prepared in 5% (wt/vol) dextrose solution (Baxter Healthcare Ltd., Norfolk, UK) and diluted in media immediately before use. TOC was prepared in ethanol (EtOH). The final concentration of the vehicle in media did not exceed 0.6% (vol/vol). Drug solutions were wrapped in tinfoil, and all experiments were performed in minimal light in a specially adapted "dark" laboratory. The ambient light level of the laboratory was measured using an Iso-Tech (Model ILM350) light meter and was found to be below the limits of detection of the meter (

Laser treatment of cell cultures. After incubation with test compounds, cells were washed with phosphate-buffered saline (PBS), and the plates were irradiated on an ice block with 630 nm red light using a Diomed laser (Cambridge, UK). Using an irradiance of 120 mW/cm^sup 2^ the exposure dose delivered to the cells was 1, 10 and 25 J/cm^sup 2^. Samples that were not to be photodynamically activated were wrapped in tinfoil and sham irradiated for a period of time equal to the 25 J/cm^sup 2^ dose. A further set of controls were not sham irradiated but simply left in the dark for the duration of the laser treatment. There was no difference noted between the two sets of control.

Estimation of cell number by Hoechst 33258 staining. After freeze-thaw lysing in distilled water, a solution of Hoechst 33258 (10 �g/mL final concentration) was added to each well, and the plates were incubated for 90 min at room temperature in the dark. The samples were read on a spectrofluorimeter (Hitachi, excitation: 350 nm, emission: 460 nm).

Cell viability assays. Cell metabolic activity was measured either immediately after PDT or 24 h after PDT by the methylthiazoletetrazolium bromide (MTT) and neutral red uptake assays (NRUA). The procedure adopted for the MTT assay was that of Plumb et al. (20). In brief, after a 3 h incubation with 0.6 mg/mL MTT, the liquid was removed from the plates, which were then frozen at -20�C overnight. After defrosting the plates, 25 �L of Sorenson's glycine buffer (pH 10) was added to each well, followed by 200 �L dimethyl sulfoxide (DMSO). Absorbance of the wells was read at 570 nm in a 96-well platereader (Dynatech). Neutral red dye was prepared as a 4% solution in distilled water and diluted 1:80 in growth medium 24 h before use. After a 3 h incubation with the filtered dye, the wells were checked microscopically to ensure that no crystal formation had occurred. The medium was removed and replaced with a solution of 4% formaldehyde containing 1% CaCl^sub 2^ before being solubilized in a solution of 1% (vol/vol) glacial acetic acid in 50% (vol/vol) EtOH. Absorbance was read at 550 nm in a 96-well platereader (21).