Flow Cytometric Technique for Quantitating Cytotoxic Response to Photodynamic Therapy

Abstract

A simple flow cytometric technique for rapid measurement of multilog cytotoxic responses to photosensitization of cellular systems is described. This technique is particularly useful for cell lines with a low colony-forming efficiency, for which a nonclonogenic assay is required. The assay separates cell-sized objects from cellular debris by gating on forward scatter versus side scatter, identifies viable cells by positive calcein AM and negative ethidium homodimer-1 staining and measures cell concentration relative to an internal standard of polystyrene beads. Large numbers of cells can be analyzed rapidly. Two patient-derived small cell lung cancer cell lines, NCI-H209 and SV-E, were used to test the technique. Photordiation survival curves of the response of these cell lines to 5-aminolevulinic acid-induced protoprophyrin IX photosensitization correlated with the extent of photosensitizer accumulation. There was good agreement between the results obtained using the tritiated thymidine incorporation assay and the flow cytometric cytotoxicity assay. The technique can be used to measure cytotoxic responses to photosensitization of cell lines regardless of their plating efficiencies.

In vitro studies on the potential use of 5-aminolaevulinic acid-mediated photodynamic therapy for gynaecological tumours

“…It has been used for endometrial ablation and to prevent embryonic implantation in laboratory animals (Yang et al, 1993(Yang et al, , 1994(Yang et al, , 1995Wyss et al, 1994 (Falcon). Accumulation of protoporphyrin IX (PpIX) fluorescence induced by ALA was assessed by an EPICS Elite flow cytometer (Coulter Electronics, Burlington, Ontario, Canada) as previously described (Campbell et al, 1996). This method allowed the individual examination of the fluorescence emitted by several thousands of cells (n>5000).…”

“…The autofluorescence of the control sample was subtracted from the mean fluorescence intensity of each sample and these values were expressed in arbitrary fluorescence units. The data were not corrected for differences in cell volume as all of the cell lines had similar volumes as assessed by the measurement of the forward scatter on the flow cytometer (Campbell et al, 1996).…”

“…The photosensitising light was focused perpendicularly on the cell culture well, which was placed on a rotating holder to ensure uniformity of irradiation (irradiance 70 + 10 mW cm-2), determined using an apertured calorimetric detector (Sciencetech 380101, Boulder, CO, USA). After a predetermined time of light exposure, cells were collected and labelled with fluorescent dyes in order to assess cell viability by flow cytometry as described elsewhere (Campbell et al, 1996) 883 localisation studies, the cells on the coverslips were further incubated for 10 min in 10 jug ml-' rhodamine 123 in RPMI (without phenol red), rinsed in RPMI without phenol red, then observed as previously described through a 525/30 nm BP filter. The no ALA and no rhodamine controls exhibited no cross-fluorescence under the experimental conditions used.…”

Rodent fibroblast model for studies of response of malignant cells to exogenous 5-aminolevulinic acid

“…Aliquots (200 µl) were placed in polystyrene flow cytometry tubes (Falcon #2058, Becton Dickinson, Lincoln Park, NJ, USA), and exposed to graded doses of photoactivating light as described previously (Campbell et al, 1996a). The fluence was approximately 70 mW cm -2 (Model 210 Power Meter, Coherent Radiation).…”

“…The basic technique was reported previously (Campbell et al, 1996a). Cultures of fibroblasts were incubated in the dark with exogenous ALA as described above.…”