The Violet Ratiometric Membrane Asymmetry Probe/Dead Cell Apoptosis Kit provides a simple and fast method for the detection of apoptosis with dead cell discrimination by flow cytometry. 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. Ratiometric probes have several advantages over traditional fluorochrome labeled reagents. The ratiometric probe is a self-calibrating absolute parameter of apoptotic transformation, which is independent of probe concentration, cell size, and instrument variation, such as fluctuations of laser intensity or sensitivity of the detectors. Given that apoptosis modifies the surface charge of the outer leaflet of the plasma membrane the violet membrane asymmetry probe F2N12S can monitor changes in membrane asymmetry that occur during apoptosis through a change in the relative intensity of the two emission bands of the dye. The F2N12S probe is combined with SYTOX(R) AADvanced dead cell stain, which is capable of passing through the cell membrane only in late apoptotic or necrotic cells allowing discrimination form early apoptotic cells. Samples can be analyzed after a 5 minute incubation at room temperature and does not require special buffers or wash steps. This kit can be paired with other reagents such as MitoProbe™ DiIC1(5) or annexin V for multiparametric analysis of apoptosis and viability. This reagent kit allow researchers to maximaize the utility of their instruments by utilizing the violet laser Each kit contains sufficient reagents for ∼100 flow cytometry tests.
