Practice question

Selective lysis of dividing cells while sparing quiescent population can be accomplished using bromodeoxyuridine mediated photosensitization strategy. Cells pulsed with BrdU during S phase incorporate halogenated analog into newly synthesized DNA. Subsequent exposure to bright visible light or near-UV in presence of DNA binding dyes such as Hoechst 33258 sensitizes BrdU-substituted DNA to photolysis where carbon-bromine bond breaks generating uracilyl radicals causing strand breaks overwhelming repair and triggering apoptosis. Non-dividing cells lacking BrdU incorporation remain resistant to same light dose. This suicide approach enriches for label-retaining slow-cycling stem cells and enables purification of G0 fraction for functional assays. MTT assay alone measures viability without selective elimination, caspase induction causes widespread death irrespective of proliferative status, and chromium-51 release assay monitors cytotoxic T lymphocyte mediated membrane lysis quantifying target cell killing but not specifically targeting S phase cells. Hence BrdU plus light provides unique tool for functional separation based on DNA replication status in heterogeneous cultures and developmental systems.