| Summary: | 碩士 === 長庚大學 === 基礎醫學研究所 === 95 === Previous work in this laboratory has demonstrated autophagic cell death in B16F10 cells that underwent photodynamic therapy (PDT) protocol and that cell-lysate so prepared in vitro can be used as a vaccine. In this study we employed flow cytometry to characterize each step in the PDT procedure and to differentiate which types of dendritic cells (DC) are the most avid scavengers of the PDT-generated vaccine. To these ends, B16F10 cells were subject to Photofrin® (P), laser irradiation (L), and starvation (S) in various combinations. In analyzing the flow data, we devised a gating scheme in the density plot where R1, R2, and R3 represent respectively whole cells, nucleus-containing cell corpses, and defecated cytoplasmic constituents. We found that coupling L to P caused a dramatic shift of R1 to R2 /R3, whereas P alone exhibited only a slight shift that could be reversed upon withdrawal of P. Since, in an accompanying work we have also demonstrated the reversible effect of P on autophagic vacuole formation (Li, 2007), and that cell size reduction is shown to be a hallmark of autophagy (Hosokawa et al, 2006), we conclude that flow cytometry can be used as a tool in studying autophagy and that Photofrin® is an autophagy inducer. Finally, by coculturing the PDT-generated B16F10 cell-lysate and spleen-derived DC, we demonstrated that CD8+ DC had a better capacity than CD8- DC in taking up the vaccine.
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