Cells were incubated for 5 days under standard culture conditions and then stained with dilutions of ViViD dye (20, 10, 5, 2.5, 1.25 and 0.625ug/ml). In summary, amine reactive viability dyes can be a powerful substitute for more traditional viability dyes and can be used in a variety of panel designs.įigure 3 is a representative titration of the ViViD dye used in staining panels. In ICS (Intracytoplasmic Cytokine Staining) and peptide-MHC Class I (“tetramer”) assays, ( Chattopadhyay et al., 2008 Perfetto et al., 2006b), where detection of rare events is critical, non-specific binding of mAB-conjugated by dead cells may result in significant overestimation of the proportion of antigen-specific cells. Such samples often include large numbers of dead cells, which can bind m-Ab conjugates non-specifically. The utility of amine reactive dyes is best demonstrated in the context of staining panels, which employ various combinations of fluorochrome mAb-conjugates, using samples that have been cryopreserved and/or stimulated with antigen. In order to detect fluorescence from each emission curve the V450 detector uses only the 450/50nm band pass filter (upper insert) and the V525 detector uses a 505LP dichrolic filter combined with a 515/20nm band pass filter (lower insert). Each rectangle shows band pass filter range of detection for the PMT as shown in each insert. These dyes are excited by the violet 408nm laser indicated by the black line over the excitation curve. The instrument configuration for proper detection of two common amine reactive dyesįigure 2 shows the excitation (dotted curve) and emission (solid curve) curves of the ViViD dye (upper panel) and the Aqua Blue dye (lower panel). Additionally, it is important to note that the flow cytometer used to measure amine reactive dyes should be calibrated and standardized as described ( Perfetto et al., 2006a). Hence, ViViD is measured between 425-475nm (450/50 band pass filter), while Aqua Blue is detected between 525-575nm (505nm long pass filter + 515/20nm band pass filter). The shaded area over the emission curve shows the range of detection. For example, figure 2 shows the excitation curves (dotted lines) and the emission curves (solid lines) of ViViD and Aqua Blue, which can be excited by a 408nm violet laser (vertical black line). To measure these two dyes, it is critical to determine the correct filter configuration for detecting signal from each dye on the flow cytometer. Notably, the reaction is irreversible therefore, when cells are fixed and permeabilized (as with intracellular staining procedures), the bound dye remains associated with the dead cells (unlike other viability dyes). Live cells exclude these dyes because their cell membranes are intact, and free dye is washed away after staining ( Figure 1). These dyes differ from other viability markers because they react with free amines in the cytoplasm. Two of the commonly used amine reactive dyes, ViViD and Aqua Blue are described in this protocol. There are eight LIVE/DEAD® fixable dead cells stains available in the market today, covering most of the visible and UV spectrum. Amine reactive viability dyes offer an innovative alternative to traditional viability markers.
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