Erythrosine-Based PDT of Oral Epithelial Cells Figure 8. Caspase 3/7 Activity Assay. Caspase 3/7 activation in response to PDT was estimated at four different recovery time points post-PDT (0, 2, 4 and 24 hr) in DOK (A) and H357 cells (B) at 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. In order to bring the data sets of both DOK and H357 cells to the same scale, the luminometer arbitrary values for DOK cells were divided by 10. The values are means of three replicate determinations 6 S.D. Statistical analysis was done via student’s t-test with significance set at P,0.05 (* indicates statistical comparison as indicated by the lines). doi:10.1371/journal.pone.0034475.g008 in each sample was determined by comparison of the sample’s fluorescence with that of a standard erythrosine solution in 0.1 M NaOH. The amount of protein in each sample was also determined using the BCA (bicinchoninic acid) Protein Assay Kit (Pierce, UK), as per the manufacturer’s instructions. All erythrosine uptake experiments were carried out in quadruplicate for each treated and control sample. Erythrosine uptake was expressed as mg erythrosine/mg cell protein. PDT based Cell Killing DOK and H357 cells were plated in 96-well plates (56103 cells/ well) 24 hours before the treatment. Duplicate wells were incubated with erythrosine 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) for 30 min. Cell monolay- ers were then washed (63 with PBS) and subjected to PDT using 0, 2 2 2 30 (40.86 J/cm ), 60 (81.72 J/cm ) and 90 (122.58 J/cm ) min irradiation times (fluences). After irradiation, plates were incubated at 37uC for 12 hr before quantification of cell viability using the CellTiter 96HAQueous One Solution Cell Proliferation Assay (Promega, USA) as per the manufacturer’s instructions. Formazan formed by viable cells was quantified by reading the plates at 490 nm using a Microplate Reader (Dynex Technologies Ltd., UK). Cell kill following PDT were converted into percentage cell kill using the following formulae: PLoS ONE | www.plosone.org 10 April 2012 | Volume 7 | Issue 4 | e34475