Erythrosine-Based PDT of Oral Epithelial Cells Figure 7. Confocal Laser Scanning Microscopy (CLSM) Analysis. CLSM images of DOK (A) and H357 (B) cells at different recovery time points post-PDT (2, 4 and 24 h) were taken for both low (71.03 mM) and high (1136.50 mM) doses of erythrosine. For both doses, PDT irradiation was done with a fluence of 122.58 J/cm2. Green (SYTO9) fluorescence represents both live and dead cells while red (Propidium Iodide) fluorescence stains the dead cells alone. The arrow in the DOK cells (A) indicates the presence of apoptotic bodies whereas the arrow in the H357 cells (B) points to a group of necrotic cells. doi:10.1371/journal.pone.0034475.g007 (ECACC). DOK (pre-malignant; ECACC No. 94122104) [47] and H357 (malignant; ECACC No. 06092004) [48] cells were derived from dysplastic oral keratinocytes and squamous cell carcinoma (SCC) of the tongue respectively. Both cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma- Aldrich, UK) supplemented with 10% Fetal Calf Serum (Sigma- Aldrich, UK), 50 U/ml of Penicillin-Streptomycin (Sigma-Al- drich, UK) and 5 mg/ml of Hydrocortisone (Sigma-Aldrich, UK) and maintained at 37uC with 5% CO2 atmosphere. Preparation of Erythrosine Erythrosine B sodium salt (Sigma-Aldrich, UK) was dissolved in DMEM to give a final concentration of 1 mg/ml. This solution was then sterilized using a 0.2 mm filter and serially diluted to produce photosensitizer concentrations of 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml and 0.0625 mg/ml for use in the following studies. Light Source:- A 400W Tungsten filament lamp with a fluence rate of 22.7 mWcm22 in the 500–550 nm wavelength range was used for illumination of samples. A water tank was used to remove infra- red. Samples were placed under the water tank at a distance of 30 cm from the lamp during irradiation. The irradiance was calculated under the water tank at a distance of 30 cm from the lamp (treatment location). Throughout this study, control samples are cells incubated with erythrosine B but not irradiated (i.e. untreated samples). Erythrosine Uptake Analysis DOK and H357 cells were seeded in 6-well plates (104 cells/ well) and returned to the incubator overnight. The cells were then incubated with erythrosine at concentrations of 0.0625 mg/ml (71.03 mM), 0.125 mg/ml (142.06 mM), 0.25 mg/ml (284.13 mM), 0.5 mg/ml (568.27 mM) and 1 mg/ml (1136.5 mM) at 37uC or at 4uC for 30 min. The cells were then washed (63) with Dulbecco’s Phosphate Buffer Saline (PBS), incubated with 0.1 M NaOH (1 ml, for 1–2 min) and harvested using a cell scraper. The samples were then diluted (1:10) with 0.1 M NaOH followed by determination of fluorescence intensity using an excitation (550 nm) and emission (540 nm) wavelength in the Microplate Reader (Dynex Technologies Ltd, UK). Erythrosine concentration PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e34475