Apoptosis is carefully regulated and essential part of normal tissue development and homeostasis. Regulatory changes in the apoptotic pathway have been implicated in many diseases; most notable are many types of cancers and autoimmune disorders. Normal cells exhibit a remarkable asymmetry in lipid distribution between the outer and inner cell membranes characterized by phosphatidyl-serine (PS) and phosphatidylethanolamine (PE) normally located on the inner leaflet of the cell membrane. During apoptosis translocation of PE and PS to the external cellular environment facilitates recognition and elimination of these cells by macrophages. The Violet Ratiometric Membrane Asymmetry Probe, 4′-N, N-diethylamino-6-(N, N, N-dodecyl-methylamino-sulfopropyl)-methyl-3-hydroxyflavone (F2N12S), is a novel violet excitable dye for the detection of membrane asymmetry changes during apoptosis. The dye exhibits an excited-state intramolecular proton transfer (ESIPT) reaction resulting in a dual fluorescence with two emission bands corresponding to 530 nm and 585 nm, producing a two-color ratiometric response to variations in surface charge. This ratiometric probe is a self-calibrating absolute parameter of apoptotic transformation, which is independent of probe concentration, cell size, and instrument variation. The two-color ratiometric response of the F2N12S reagent will detect apoptotic cells in samples that vary in cell concentration by as much as 100 fold. The Violet Ratiometric Membrane Asymmetry Probe rapidly targets the plasma membrane requiring only a 5 minute room temperature incubation without additional buffers or wash steps reducing the chance of cell death that can occur from prolonged sample processing. The Violet Ratiometric Membrane Asymmetry Probe will work in both suspension and adherent cell lines and its efficacy is confirmed by caspase and mitochondrial apoptosis markers. Annexin V staining produced a larger population of apoptotic cells during early time points in both suspension and adherent cell lines that do not correlate with caspase and mitochondrial apoptosis markers. The violet 405 nm excitation of F2N12S expands the capability of multi-parameter apoptosis assays to study the apoptotic process while reducing or eliminating the need to create complex compensation controls by taking advantage of the time and spectral separation a violet-excitable reagent provides.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3976.