% Cell Kill~100| (A490 Control Cells{A490 Treated Cells)=A490 Control Cells Sub-cellular Localization Analysis by CLSM DOK and H357 cells were seeded in Sterilin Petri-dish. Following attachment they were incubated with a concentration of 1136.5 mM erythrosine at 37uC for 30 min in the dark. For organelle localization comparison, cells were either co-incubated with 100 nM LysoTracker Green (Invitrogen) or 100 nM MitoTracker Green (Invitrogen). Samples were then observed directly using a Leica TCS SP2 Confocal Laser Scanning Microscope (CLSM). All the images were analyzed via ImageJ software (W. S. Rasbaud, Image J, NIH, Bethesda, MD, http:// rsb.info.nih.gov/ij/). Co-localization of either LysoTracker or MitoTracker with erythrosine was (statistically) evaluated through the agency of co-localization frequency scatter plots, Pearson’s coefficient and Mander’s coefficient as explained elsewhere [29]. Mitochondrial Trans-Membrane Potential (DYm) Analysis via Flow Cytometry DOK and H357 cells were seeded in 21.5 cm2 cell culture plates as described above. Following incubation with erythrosine concentrations of 0.0625 mg/ml (71.03 mM) and 1 mg/ml (1136.5 mM) for 30 min, the cells were irradiated for 90 min 2 (122.58 J/cm ). Cells were then trypsinised at three different recovery time-points (0, 2 and 24 h) after irradiation followed by cell pelleting and washing with PBS. The cell pellet was then resuspended in PBS containing 5 mg/ml of Rhodamine B followed by incubation for 30 min at 37uC. Subsequently, the cells were analyzed using the FACS Calibur Flow Cytometry (BD Biosci- ences, USA). The results were processed using CellQuest Software (BD Biosciences, USA). For each sample, 10,000 events were collected. The results generated by CellQuest software were processed using the software, WinMDI v.2.9 (http://facs.scripps. edu/software.html). Here, the intensity of rhodamine B fluores- cence was taken as direct indication of overall mitochondrial membrane potential (DYm) for a particular cell population. All the calibrations of the flow cytometer were set-up by Dr. Gareth Howell (Research Fellow, Institute of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, UK). Cell Death Morphology Analysis using Scanning Electron Microscopy (SEM) DOK and H357 cells were prepared as stated above in 21.5 cm2 cell culture plates for two different erythrosine References 1. British Dental Association (2000) Opportunistic Oral Cancer Screening: A management strategy for Dental Practice. Occasional Paper (British Dental Association) No. 6. 2. Argiris A, Eng C (2004) Epidemiology, staging and screening of head and neck cancer. In: Brockstein B, Masters G, eds. Head and Neck Cancer. New York: Kluwer Academic Publishers. pp 14–60. 3. Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA Cancer J Clin 55: 74–108. 4. Konopka K, Goslinski T (2007) Photodynamic therapy in dentistry. J Dent Res 86: 694–707. 5. Garg AD, Krysko DV, Vandenabeele P, Agostinis P (2011) DAMPs and PDT- mediated photo-oxidative stress: exploring the unknown. Photochem Photobiol Sci 10: 670–680. 6. Garg AD, Nowis D, Golab J, Agostinis P (2010) Photodynamic therapy: illuminating the road from cell death towards anti-tumour immunity. Apoptosis 15: 1050–1071. Erythrosine-Based PDT of Oral Epithelial Cells concentrations 20.0625 mg/ml (71.03 mM) and 1 mg/ml (1136.5 mM). Following incubation with erythrosine, the cells were irradiated for 90 min (122.58 J/cm2) followed by fixation for 1 hr using 2.5% gluteraldehyde in PBS (pH 7.4) at three different recovery time-points (0,2 and 24 h) after irradiation. Subsequently, the cells were washed with PBS (36) to ensure complete gluteraldehyde removal. Samples were then dehydrated through a graded ethyl alcohol series. Following dehydration, the plates were cut into suitable pieces, fixed to aluminum stubs and sputter- coated with gold using an Automatic Sputter Coater (Agar Scientific, UK). The samples were then visualized directly using a Hitachi VP-SEM S-3400N Scanning Electron Microscope. Cell Death Morphology Analysis using CLSM DOK and H357 cells were prepared as stated above in 21.5 cm2 cell culture plates for two different erythrosine concentrations 20.0625 mg/ml (71.03 mM) and 1 mg/ml (1136.5 mM) and subjected to PDT 290 min irradiation 2 (122.58 J/cm ). Following PDT, the cells were stained using SYT09 stain (green fluorescence; excitation l- 420 nm, emission l-580 nm) which stains the nucleic acid in both live/dead cells and Propidium Iodide (PI) (red fluorescence; excitation l- 420 nm, emission l-580 nm) which stains the nucleic acids of dead cells alone; at recovery time points of 2, 4 and 24 h following irradiation. Cells were then observed immediately using a Leica TCS SP2 CLSM. Caspase Activity Assay Samples were prepared as stated above in 96-well plate for two different erythrosine concentrations 20.0625 mg/ml (71.03 mM) and 1 mg/ml (1136.5 mM) and subjected to PDT 290 min 2 irradiation (122.58 J/cm ). Following PDT, the Caspase activity assay was performed using the Caspase-Glo3/7 assay (Promega, Madison, USA) kit at four different recovery time point (0, 2, 4 and 24 h) as per the manufacturer’s instruction. Then the plates were observed for the luminescence signal in the luminometer (BMG Labtech Fluostar Optima, Germany). Calibrations of the luminometer were performed by Dr. Carsten Zoothner (Techni- cian, Institute of Molecular and Cellular Biology, University of Leeds, UK). Author Contributions Conceived and designed the experiments: SW ADG MB MA WB. Performed the experiments: SW ADG MB MA WB. Analyzed the data: SW ADG MB MA WB. Contributed reagents/materials/analysis tools: SW WB. Wrote the paper: SW ADG MB MA WB. 7. Agostinis P, Berg K, Cengel KA, Foster TH, Girotti AW, et al. (2011) Photodynamic therapy of cancer: An update. CA Cancer J Clin 61: 250–281. 8. Garg AD, Krysko DV, Vandenabeele P, Agostinis P (2012) Hypericin-based photodynamic therapy induces surface exposure of damage-associated molecular patterns like HSP70 and calreticulin. Cancer Immunol Immunother 61: 215–221. 9. Garg AD, Krysko DV, Verfaillie T, Kaczmarek A, Ferreira GB, et al. (2012) A novel pathway combining calreticulin exposure and ATP secretion in immunogenic cancer cell death. EMBO J 31: 1062–1079. 10. Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, et al. (1998) Photodynamic therapy. J Natl Cancer Inst 90: 889–905. 11. Estevez JP, Ascencio M, Colin P, Farine MO, Collinet P, et al. (2010) Continuous or fractionated photodynamic therapy? Comparison of three PDT schemes for ovarian peritoneal micrometastasis treatment in a rat model. Photodiagnosis and Photodynamic Therapy 7: 251–257. PLoS ONE | www.plosone.org 11 April 2012 | Volume 7 | Issue 4 | e34475