In Vitro PDT Dose-Response Assays

A431 squamous carcinoma cells were obtained from American Type Culture Collection (ATCC, Rockville, MD) and grown in DMEM (HyClone, Logan, Utah) supplemented with 10% FBS (HyClone), 100 IU/ml penicillin (Life Technologies, Carlsbad, CA), and 100 μg/ml streptomycin (Life Technologies). Cell cultures were maintained at 378C in a humid atmosphere of 95% air and 5% CO₂ and harvested for treatment assessment studies either in multiwell plates (for in vitro studies) or for murine tumor implantation as described below. For in vitro dose response experiments 96 well plates were used with three replicate wells for each treatment condition and control group. For cell kill area measurements cultures were plated in 35 mm dishes with each treatment condition in triplicate. In both cases cultures were photosensitized using 2.0 mM 5-ALA (Sigma–Aldrich) in media for 4 hours prior to light activation. Light was delivered at 30 mW/cm² either continuously or fractionated as indicated by mounting the fiber optic vertically underneath the cell culture vessel as in previous in vitro PDT experiments [14 ,16 (link)].